Methods for up-regulating antigen expression in tumors

ABSTRACT

The invention provides methods of modulating tumor antigen associated (TAA) expression, and methods of modulating TAA expression in order to treat a tumor. More particularly, the invention provides methods of increasing an immune response against a tumor cell. Methods include administering to a subject with a tumor an amount of IFN-β receptor agonist and tumor associated antigen (TAA) sufficient to increase an immune response against the tumor cell.

RELATED APPLICATIONS

This application claims priority to U.S. Application Ser. No.60/407,492, filed Aug. 29, 2002.

FIELD OF THE INVENTION

The invention relates to modulating tumor antigen associated (TAA)expression, and more particularly to methods of modulating TAAexpression in order to treat a tumor.

BACKGROUND

Many solid tumors are presently known to involve the infiltration ofautologous lymphocytes. These autologous lymphocytes, known astumor-infiltrating lymphocytes (TIL), have been shown to recognizespecific antigens expressed by cells of the solid tumor. Expression ofsuch tumor-associated antigens (TAAs) in combination with appropriateaccessory signals leads to a specific cytolytic (cytotoxic) reactivityof the TILs toward the solid tumors. In addition, antibodies that canrecognize similar and unique antigens have also been shown to bindselectively to and facilitate killing of tumor cells.

Several tumor antigens have been identified in association with avariety of tumors (Boon, et al. (1994). Ann Rev Immunol, 12:337;Kawakami, et al. (1994). Proc Natl Acad Sci USA, 91:3515; and Bakker, etal. (1994). J Exp Med, 179:1005). In addition to the identification ofTAAs, immunodominant epitopes recognized by TILs have also beendescribed for widely-expressed lineage-specific antigens, for example,the HLA-A2-restricted Melan-A/MART-1 in melanomas (Sensi, et al.,(1995). Proc Natl Acad Sci USA, 92:5674; and Kawakami, et al., (1994). JExp Med, 180:347).

Although there is mounting evidence that it is possible to induce cellmediated immunity against autologous melanomas, clinical immunotherapystrategies (Kradin, et al. Cancer Immunol. Immunother. (1987). 24:76);Kradin, et al. Lancet, (1989). 1:577; Rosenberg et al., (1987). N. Eng.J. Med., (1988). 25:1676; Dillman, et al. (1991). Cancer, 68:1; Gattoni,et al., (1966). Semin. Oncol, 23:754; and Kan-Mitchell, et al. (1993).Cancer Immunol. Immunother., 37:15), have failed to achieve routineefficacy. This failure has been due, at least in part, to the ability oftumors to evade immune destruction (Becker, et al., (1993). Int.Immunol., 5:1501; Jager, et al. (1997). Int. J. Cancer, 71:142; Macurer,et al., (1996). J. Clin. Invest., 98:1633; and Marincola, et al.,(1996). J. Immunother. Emphasis Tumor Immunol., 9:192).

SUMMARY

The invention provides methods of increasing an immune response againsta tumor cell. In one embodiment, a method includes administering to asubject with a tumor an amount of IFN-β receptor agonist and tumorassociated antigen (TAA) sufficient to increase an immune responseagainst the tumor cell. An immune response includes cell-mediated orhumoral immune responses.

Also provided are methods of inhibiting silencing of a tumor associatedantigen (TAA), and methods of increasing expression of a tumorassociated antigen (TAA). In one embodiment, a method includesadministering to a subject with a tumor an amount of IFN-β receptoragonist sufficient to inhibit silencing of the tumor associated antigen(TAA). In one aspect, the subject has been administered a tumorassociated antigen (TAA) prior to, substantially contemporaneously withor following IFN-β receptor agonist administration. In anotherembodiment, a method includes contacting a cell capable of expressing aTAA with a compound that modulates an activity of an NFAT-motif bindingprotein in an amount sufficient to increase expression of a tumorassociated antigen (TAA) of the cell.

Further provided are methods of treating a tumor. In one embodiment, amethod includes administering to a subject with a tumor an amount ofIFN-β receptor agonist and tumor associated antigen (TAA) sufficient totreat the tumor. In another embodiment, a method includes administeringto a subject with a tumor an amount of IFN-β receptor agonist and anantibody or a cell that produces an antibody that specifically binds toa tumor associated antigen (TAA) sufficient to treat the tumor. In yetanother embodiment, a method includes administering to a subject with atumor an amount of IFN-β receptor agonist and an immune cell thatinteracts with a tumor cell sufficient to treat the tumor.

Additionally provided are methods of treating a subject having or atrisk of having a tumor. In one embodiment, a method includesadministering to the subject an amount of IFN-β receptor agonist andtumor associated antigen (TAA) sufficient to treat the subject. Inanother embodiment, a method includes administering to the subject anamount of IFN-β receptor agonist and an antibody or a cell that producesan antibody that specifically binds to a tumor associated antigen (TAA)sufficient to treat the subject. In yet another embodiment, a methodincludes administering to the subject an amount of IFN-β receptoragonist and an immune cell that interacts with a tumor cell sufficientto treat the subject.

Moreover provided are methods of increasing effectiveness of ananti-tumor therapy. In one embodiment, a method includes administeringto a subject that is undergoing or has undergone tumor therapy, anamount of IFN-β receptor agonist and tumor associated antigen (TAA)sufficient to increase effectiveness of the anti-tumor therapy. Inanother embodiment, a method includes administering to a subject that isundergoing or has undergone tumor therapy, an amount of IFN-β receptoragonist and an antibody or a cell that produces an antibody thatspecifically binds to a tumor associated antigen (TAA) sufficient toincrease effectiveness of the anti-tumor therapy. In yet anotherembodiment, a method includes administering to a subject that isundergoing or has undergone tumor therapy, an amount of IFN-β receptoragonist and an immune cell that interacts with a tumor cell sufficientto increase effectiveness of the anti-tumor therapy.

IFN-β receptor agonists useful in the invention include, for example,IFN-β, an IFN-β mimic, or an IFN-β receptor antibody. Compounds andagents useful in the invention also include molecules having similaractivity as IFN-β (e.g., having TAA-inducing activity).

Compounds that modulate an activity of an NFAT-motif binding proteininclude calcium flux modulators (e.g., ionomycin and verapimil), VIVIT,gossypol, an N-substituted benzamide, rapamycin, aquinazoline-2,4-dione, 1-3, a pyrrolo[3,4-d]pyrimidine-2,4-dione, 4-8,1alpha,25-dihydroxyvitamin D3, FK506, FK520, cyclosporin,3,5-Bis(trifluoromethyl)pyrazoles, dithiocarbamates, Vasoactiveintestinal peptide (VIP) and pituitary adenylate cyclase-activatingpolypeptide (PACAP), Carboxyamidotriazole, Morphine, a C32-O-arylethylether derivative of ascomycin, Ascomycin macrolactam derivative SDZ ASM981, or MCIP1. Additonal compounds include, for example, an NFATantisense nucleic acid, NFAT binding protein (e.g., an antibody) or adominant negative NFAT polypeptide.

Tumors include any metastatic or non-metastatic, solid or liquid (e.g.,hematopoetic), malignant or non-malginant neplasia or cancer in anystage, e.g., a stage I, II, III, IV or V tumor. Particular embodimentsinclude a sarcoma, carcinoma, melanoma, myeloma, blastoma, lymphoma orleukemia.

Treatments provided include a therapeutic benefit, for example, reducingtumor volume, inhibiting an increase in tumor volume, stimulating tumorcell lysis or apoptosis, reducing tumor metastasis, or inhibiting tumorprogression. Treaments provided also include reducing one or moreadverse symptoms associated with the tumor, including reducing mortalityor prolonging lifespan.

Treatments provided further include administering an anti-tumor therapy(e.g., surgical resection, radiotherapy, or chemotherapy), immuneenhancing therapy (e.g., an antibody or a cell that produces an antibodythat specifically binds to a tumor associated antigen (TAA); or animmune cell that interacts with a tumor cell) and an immune-enhancingagent. Cells that produce an antibody that specifically binds to a tumorassociated antigen (TAA) include a plasma cell, B-cell, or a mammalianor non-mammalian cell transfected with a nucleic acid encoding theantibody. Immune cells that interact with a tumor cell include T cell,NK cell, LAK cell, monocyte or macrophage, including cells pre-selectedto bind to an antigen expressed by the tumor.

Methods of identifying an agent that increases expression of a melanomatumor associated antigen (TAA) are additionally provided. In oneembodiment, a method includes contacting a cell capable of expressing amelanoma TAA (e.g., a melanoma cell) with a test agent; measuring theamount of TAA (e.g., Melan-A/MART-1, tyrosinase, gp100/pmel 17, TRP-1,TRP-2 or MITF-M) expressed in the presence of the test agent; anddetermining whether the amount of TAA expressed is greater in thepresence than in the absence of the test agent. Increased TAA expressionidentifies the test agent as an agent that increases expression of amelanoma TAA.

TAAs modulated in accordance with the invention include, for example,antigens whose expression is increased in a tumor cell in comparison toa non-tumor cell (e.g., normal) counterpart; antigens whose expressionis approximately the same or less in tumor cell in comparison to anon-tumor cell counterpart; and antigens whose expression changes duringdevelopment, differentiation or in response to a stimulus. TAAs can bepresent on or in a cell (e.g., in the cytoplasm or nucleus or attachedto the cell surface). TAAs can be present on any tumor, for example, asarcoma, carcinoma, melanoma, myeloma, blastoma, lymphoma or a leukemia.

Exemplary TAAs include: Melan-A/MART-1, tyrosinase, gp100/pmel 17,TRP-1, TRP-2, an MITF, MITF-A, MITF-M, melanoma GP75, Annexin I, AnnexinII, adenosine deaminase-binding protein (ADAbp), PGP 9.5, Colorectalassociated antigen (CRC)—C017-1A/GA733, Ab2 BR3E4, CI17-1A/GA733, Hsp70,Hsp90, Hsp96, Hsp105, Hsp110, HSPPC-96, stress protein gp96 (a humancolorectal cancer tumor rejection antigen, Heike 2000), gp96-associatedcellular peptide, G250, Dipeptidyl peptidase IV (DPPIV), Mammaglobin,thyroglobulin, STn, Carcinoembryonic Antigen (CEA), CarcinoembryonicAntigen (CEA) epitope CAP-1, Carcinoembryonic Antigen (CEA) epitopeCAP-2, etv6, aml1, Prostate Specific Antigen (PSA), PSA epitope PSA-1,PSA epitope PSA-2, PSA epitope PSA-3, Ad5-PSA, prostate-specificmembrane antigen (PSMA), Prostatic Acid Phosphatase (PAP), Prostateepithelium-derived Ets transcription factor (PDEF),Parathyroid-hormone-related protein (PTH-rP), EGFR, PLU1, Oncofetalantigen-immature laminin receptor (OFA-iLR), MN/CA IX (CA9) (Shimizu,2003), HP59, Cytochrome oxidase 1, sp100, msa, Ran GTPase activatingprotein, a Rab-GAP (Rab GTPase-activating) protein, PARIS-1, T-cellreceptor/CD3-zeta chain, cTAGE-1, SCP-1, Glycolipid antigen-GM2, GD2 orGD3, GM3, FucosylGM1, Glycoprotein (mucin) antigens-Tn, Sialyl-Tn, TFand Mucin-1, CA125 (MUC-16), a MAGE family antigen, GAGE-1,2, BAGE,RAGE, LAGE-1, GnT-V, EP-CAM/KSA, CDK4, a MUC family antigen, HER2/neu,ErbB-2/neu, p21ras, RCAS1, α-fetoprotein, E-cadherin, α-catenin,β-catenin and γ-catenin, NeuGcGM3, Fos related antigen, Cyclophilin B,RCAS1, S2, L10a, L10a, Telomerase rt peptide, cdc27, fodrin, p120ctn,PRAME, GA733/EoCam, NY-BR-1, NY-BR-2 NY-BR-3, NY-BR-4 NY-BR-5, NY-BR-6NY-BR-7, NY-ESO-1, L19H1, MAZ, PINCH, PRAME, Prp1p/Zer1p, WT1,adenomatous polyposis coli protein (APC), PHF3, LAGE-1, SART3, SCP-1,SSX-1, SSX-2, SSX-4, TAG-72, TRAG-3, MBTAA, a Smad tumor antigen, Imp-1,HPV-16 E7, c-erbB-2, EBV-encoded nuclear antigen (EBNA)-1, Herpessimplex thymidine kinase (HSVtk), alternatively spliced isoform ofXAGE-1 (L552S), TGF beta RII frame shift mutation, BAX frame shiftmutation, or an immunogenic fragment thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D illustrate data indicating the down-regulation of antigenexpression in melanoma. MU tumor cells in A) control medium or D)control medium supplemented with human oncostatin M (OSM), or in B)supernatants from EW (containing EW produced OSM) or C) from A375 tumorcells (without OSM). Cells were stained for cytoplasmic expression ofMelan-A/MART-1 protein (A-C) or gp100 (D) and assayed by flow cytometry.Mean channel of fluorescence is shown within each.

FIGS. 2A-21 illustrate data indicating that interferon-beta (IFN-β)increases expression of melanocyte lineage antigens (Melan-A/MART-1 andGP100) in melanoma cell lines 453A, A375, MU-X, MU-89, MM96L(−), andMM96L(+). Numbers in parentheses indicate the mean channel number. Ineach set, the curve to the right (stronger fluorescence) indicatesincreased expression following IFN-β treatment.

FIG. 3 illustrates data indicating that interferon-beta overcomesdown-regulation of gp100 antigen by OSM. Control MU-89 (41.6) vs. MU-89plus OSM (29.5) vs MU-89 plus IFN-beta+OSM (63.3).

FIG. 4 illustrates data indicating that interferon-beta with5-azacytidine (AZA) or trichostatin induces high levels of antigenexpression in constitutive low antigen-expressing cells, MU-X. MU-XControl (12.8) vs. MU-X+Interferon-Beta 5,000 IU/mL (23.2) vs.MU-X+5-AZA 40 uM (39.0) vs. AZA 40 uM+Interferon-Beta 5,000 IU/mL (57.3)

FIGS. 5A and 5B illustrate the effect of A) OSM on Melanoma GeneExpression in MU-89 cells. All shown at 0.39 ng RNA/sample except GADPHand (3-Actin at 24.4 pg and TRP-1 at 15.6 ng; and B) OSM on Cytotoxic TCell Recognition of Melan-A/MART-1-expressing targets, MU.

FIG. 6 illustrates the effect of MITF-M transfection on endogenousexpression of Melan-A/MART-1. Data shown for A375 and MU-X tumor cellstransfected with MITF-M for 24 hours in the presence (10 μM) or absenceof U0126 before PCR amplification of Melan-A/MART-1. Lane 1, MITF-Mexpression plasmid; 2, Empty vector control; 3, Transfection reagentsonly; 4, Untransfected control.

FIG. 7 illustrates increased killing by T lymphocytes following IFN-βtreatment of melanoma cells.

FIG. 8 illustrates an exemplary reporter construct to identify compoundshaving an activity of IFN-β. GFP reporter gene driven by the 1176-bpMelan-A/MART-1 promoter.

FIG. 9 illustrates augmentation of GFP fluorescence following exposureof transfected cells to IFN-β.

DESCRIPTION

The invention is based at least in part on the finding thatinterferon-beta (IFN-β) increases expression of one or more tumorassociated antigens (TAAs). Increasing expression of a tumor associatedantigen of a cell, such as a tumor cell, increases recognition by theimmune system. Thus, treating a tumor cell or tumor cell population withIFN-β, an IFN-β receptor agonist, or a compound or agent having aTAA-inducing activity as IFN-β, can increase antigenicity of tumorcells, thereby increasing recognition of tumor cells by T lymphocytesand antibodies. Consequently, the immune system is more likely to targetthe tumor cell(s) for destruction.

TAA expression on a cell can be increased with IFN-β, an IFN-β receptoragonist, or a compound or agent having similar activity as IFN-β (hasTAA-inducing activity). IFN-β, an IFN-β receptor agonist, or a compoundor agent having a TAA-inducing activity as IFN-β can be combined withone or more other compounds, agents, treatments or therapies having ananti-tumor effect. Thus, IFN-β, an IFN-β receptor agonist, or a compoundor agent having a TAA-inducing activity as IFN-β can be used incombination with any other anti-tumor treatment or therapeutic protocol.For example, IFN-β, an IFN-β receptor agonist, or a compound or agenthaving a TAA-inducing activity as IFN-β can be combined with anytreatment that increases an immune response against a tumor, therebyinhibiting tumor cell growth.

Thus, in accordance with the invention, methods of increasing an immuneresponse against a tumor cell are provided. In one embodiment, a methodincludes administering to a subject having a tumor an amount of IFN-βreceptor agonist and a tumor associated antigen (TAA) sufficient toincrease an immune response against the tumor cell. In various aspects,an IFN-β receptor agonist comprises IFN-β, an IFN-β mimic (e.g., variantor modified form), or an IFN-β receptor antibody. In additional aspects,the immune response is cell-mediated or humoral. In further aspects, TAAis adminstered as full length or antigenic fragments, or with cells(e.g., cells that express TAA, such as tumor cells).

As used herein, “immune response” refers to a cell mediated or humoral(antibody mediated) response known in the art to be a function of theimmune system. Stimulating, inducing or up-regulating an immune responsemeans that either a cell mediated or humoral immune response isincreased or triggered. For example, a melanoma TAA (e.g., an epitope ofMelan-A/MART-1) can be administered and a CTL (cytotoxic T-lymphocyte)response to this antigen in a subject with metastatic melanoma elicited.

As used herein, an “IFN-β receptor agonist” means a molecule that bindsto IFN-alpha/beta receptor (IFNAR), subunits IFNAR-1 or IFNAR-2, andwhich elicits a response typical of IFN-β. An exemplary responseincludes increasing TAA expression, i.e., a TAA inducing activity.

The invention also provides methods of increasing tumor associatedantigen expression on a cell (e.g., a tumor cell). In one embodiment, amethod includes administering to a subject having a tumor an amount ofIFN-β receptor agonist and a tumor associated antigen (TAA) sufficientto increase tumor associated antigen expression on a tumor cell. In oneaspect, an immune enhancing agent (e.g., lymphocytes or antibody orantibody expressing cells specific for TAA expressed by the tumor) isadminstered prior to, substantially contemporaneously with or followingadministration of IFN-β receptor agonist or a tumor associated antigen(TAA).

As used herein, the term “tumor associated antigen” or “TAA” refers toan antigen capable of expression by a tumor cell, or on cells of thesame lineage as the tumor. The TAA in tumor may be expressed in amountsgreater than normal relative to a non-tumor (normal) cell counterpart,or may be expressed at similar levels, or at levels less than normalcell counterparts, particularly if the gene encoding the TAA isdown-modulated in the tumor cell.

Tumor associated antigens are antigenic molecules whose expressionfacilitates interaction of immune cells or immune molecules (e.g.antibodies) with tumor cells. TAAs are molecules or portions of themolecules that immune targeting molecules (i.e. receptors on immunecells and antibodies) bind. As discussed, TAAs may be present in or onnormal cells; tumor TAA expression may but need not deviate from normal(non-tumor) counterpart cells (e.g., a normal cell not expressing TAA,expressing less of the TAA than a tumor cell, or expressing the same ormore TAA than tumor.)

A tumor associated antigen can be expressed during an earlierdevelopmental or different differentiation stage of the cell; afterprogressing through the developmental stage, expression of the TAA istypically altered. For example, a melanoma differentiation associated(mda) gene displaying enhanced or suppressed expression during growthinhibition and differentiation, such as MAGE and Melan-A/MART-1. Asdisclosed herein, TAA expression can also be induced or increased inresponse to a stimulus (e.g., IFN-β). In addition, kinase inhibitors canup-regulate TAA expression (Englaro et al. (1998). J Biol Chem 273:9966)of Melan-A/MART-1, gp100, tyrosinase, TRP-1 and TRP-2 on melanomas andTAA expression has been reported to up-regulated of by interferon-gamma(Gudagni et al. (1996). In Vivo 7:591). Tumor cell expression of one ormore TAA's that are atypical for the cell is presumably due to aberrantgene regulation of the TAA.

Although not wishing to be bound by any theory, down-regulation of TAAsis thought to contribute to tumor cell escape from immune detection.Oncostatin M (OSM) (Durda et al. (2003). Mol Cancer Res 1:411) and IFN-γ(Le Poole et al (2002) Am J Pathol 160:521) can down-modulateMelan-A/MART-1 expression on melanoma cells.

Specific non-limiting examples of TAAs whose expression can be increasedor induced in accordance with the invention include, for melanoma,tumor-associated testis-specific antigen (e.g., MAGE, BAGE, and GAGE),melanocyte differentiation antigen (e.g., tyrosinase, Melan-A/MART-1), amutated or aberrantly expressed molecule (e.g., CDK4, MUM-1,beta-catenin), gp100/pmel 17, TRP-1, TRP-2, an MITF, MITF-A and MITF-M(King, et al. (1999). Am J Pathol 155:731). Additional specific examplesof TAAs expressed by tumors include melanoma GP75, Annexin I, AnnexinII, adenosine deaminase-binding protein (ADAbp), PGP 9.5 (Rode, et al.(1985). Histopathology 9:147), colorectal associated antigen(CRC)—C017-1A/GA733, Ab2 BR3E4, CI17-1A/GA733, Hsp70 (Chen, et al.(2002). Immunol Lett 84:81), Hsp90, Hsp96, Hsp105, Hsp110, HSPPC-96(Caudill, M. M. and Z. Li (2001). Expert Opin Biol Ther 1:539), stressprotein gp96 (a human colorectal cancer tumor rejection antigen, Heikeet al. (2000). Int J Can 86:489), gp96-associated cellular peptides,G250, Dipeptidyl peptidase IV (DPPIV), Mammaglobin (Tanaka, et al.(2003). Surgery 133:74), thyroglobulin, STn (Morse, M. A. (2000). CurrOpin Mol Ther 2:453), Carcinoembryonic Antigen (CEA), CarcinoembryonicAntigen (CEA) epitope CAP-1, Carcinoembryonic Antigen (CEA) epitopeCAP-2, etv6, aml1, Prostate Specific Antigen (PSA), PSA epitope PSA-1,PSA epitope PSA-2, PSA epitope PSA-3 (Correale, et al. (1998). J Immunol161:3186) (Roehrbom, et al. (1996). Urology 47:59), Ad5-PSA,prostate-specific membrane antigen (PSMA), Prostatic Acid Phosphatase(PAP), Prostate epithelium-derived Ets transcription factor (PDEF),Parathyroid-hormone-related protein (PTH-rP), EGFR (Plunkett, et al.(2001). J Mammary Gland Biol Neoplasia 6:467), PLU1 (Plunkett, et al.(2001). J. Mammary Gland Biol Neoplasia 6:467), Oncofetalantigen-immature laminin receptor (OFA-iLR), MN/CA IX (CA9) (Shimizu etal., (2003). Oncol. Rep. September-October; 10:1307), HP59, Cytochromeoxidase 1, sp100, msa (Devine, et al. (1991). Cancer Res 51:5826), RanGTPase activating protein, a Rab-GAP (Rab GTPase-activating) protein,PARIS-1 (Zhou, et al. (2002). Biochem Biophys Res Commun 290:830),T-cell receptor/CD3-zeta chain, cTAGE-1, SCP-1, Glycolipid antigen-GM2,GD2 or GD3, GM3 (Bada, et al. (2002). Hum Exp Toxicol 21:263),FucosylGM1, Glycoprotein (mucin) antigens-Tn, Sialyl-Tn (Lundin, et al.(1999). Oncology 57:70), TF and Mucin-1 (Mukherjee, et al. (2003). JImmunother 26:47), CA125 (MUC-16) (Reinartz, et al. (2003). Cancer Res63:3234), a MAGE family antigen, GAGE-1,2, BAGE, RAGE, LAGE-1(Eichmuller, et al. (2003). Int J Cancer 104:482) (Chen, et al. (1998).Proc Natl Acad Sci USA 95:6919), GnT-V (Murata, et al. (2001). Dis ColonRectum 44:A2-A4), MUM-1 (Kawakami, et al. (1996). Keio J Med 45:100),EP-CAM/KSA (Ullenhag, et al. (2003). Clin Cancer Res 9:2447), CDK4, aMUC family antigen, HER2/neu, ErbB-2/neu, p21ras, RCAS1, α-fetoprotein,E-cadherin, α-catenin, β-catenin and γ-catenin, NeuGcGM3 (Carr, et al.(2003). J Clin Oncol 21:1015), Fos related antigen (Luo, et al. (2003).Proc Natl Acad Sci USA 100:8850), Cyclophilin B (Tamura, et al. (2001).Jpn J Cancer Res 92:762), RCAS1, S2 (Koga, et al. (2003). TissueAntigens 61:136), L10a (Koga, et al. (2003). supra), L10a, Telomerase rtpeptide (Wang, et al. (2001). Oncogene 20:7699), cdc27, fodrin, p120ctn,PRAME, GA733/EoCam (Ross, et al. (1986). Biochem Biophys Res Commun135:297), NY-BR-1, NY-BR-2 NY-BR-3, NY-BR-4 NY-BR-5, NY-BR-6 NY-BR-7(Jager, et al. (2001). Cancer Res 61:2055), NY-ESO-1, L19H1, MAZ(Daheron, et al. (1998). Leukemia 12:326), PINCH (Greiner, et al.(2000). Exp Hematol 28:1413), PRAME (Ikeda, et al. (1997). Immunity6:199), Prp1p/Zer1p, WT1 (Oka, et al. (2002). Curr Cancer Drug Targets2:45), adenomatous polyposis coli protein (APC), PHF3, LAGE-1, SART3(Miyagi, et al. (2001). Clin Cancer Res 7:3950), SCP-1 (Jager, et al.(2002). Cancer Immun 2:5), SSX-1, SSX-2, SSX-4, TAG-72 (Buchsbaum, etal. (1999). Clin Cancer Res 5(10 Suppl): 3048s-3055s), TRAG-3 (Chen, etal. (2002). Lung Cancer 38:101), MBTAA (Basu, et al. (2003). Int JCancer 105:377), a Smad tumor antigen, lmp-1, HPV-16 E7, c-erbB-2,EBV-encoded nuclear antigen (EBNA)-1, Herpes simplex thymidine kinase(HSVtk), alternatively spliced isoform of XAGE-1 (L552S; Wang, (2001).Oncogene 20:7699), TGF beta RH frame shift mutation (Saeterdal, et al.(2001). Proc Natl Acad Sci USA 98:13255), BAX frame shift mutation(Saeterdal, et al. (2001). Proc Natl Acad Sci USA 98:13255).

Immunogenic fragments (subsequences, including antigenic peptides thatcan be targeted) of TAAs are also included. In addition, variants andmodified forms of TAA capable of eliciting, increasing or stimulating animmune response are also included.

TAAs can be delivered by a variety of methods. For example, whenadministering one or more TAAs with IFN-β, an IFN-β receptor agonist, ora compound or agent having a TAA-inducing activity as IFN-β, the TAA canbe formulated to be presented to the immune system to stimulate animmune response towards the TAA. Thus, a TAA or antigenic fragment, ortumor or other cell having TAA can be adminstered in vivo. Tumor cellsexpressing TAA can optionally be treated ex vivo (e.g., with IFN-β, anIFN-β receptor agonist, or a compound or agent having similar activityas IFN-β) and transfused into a patient during therapy. Any agent thatenhances antigen expression or antigenicity of the tumor can be used totreat the tumor in vivo or ex vivo. Tumor cell lysates or extracts, orirradiated or heat killed cells that renders them incapable of growth,but still able to induce an immune response, can also be administered.

TAAs can be delivered as peptides (Jäeger et al. (1996) Int J Cancer66:162; Jäger et al. (2000) Proc Natl Acad Sci USA 97:12198; Marchand etal. (1999) Int J. Cancer. 80:219, or as peptides in combination withadjuvants (Jäger et al. (1996). Int J Cancer 67:54; Rosenberg et al.(1998). Nat Med 4:321; Cormier et al. (1997). Cancer J Sci Am. 3:37;Wang et al. (1999). Clin Cancer Res. 5:2756).

TAAs can be delivered with other cells. For example, TAA peptides can beloaded into dendritic cells (Chen et al. (2001) Gene Ther 8:316; Fong etal. (2001). J Immunol 167:7150; Therner et al. (1999). J Exp Med190:1669; Tso et al. (2001). Cancer Res 61:7925), or loaded into otherantigen presenting cells (Pardoll (2002). Nature Rev Immunol 2:227).

Three types of DNA-based recombinant cancer vaccines have been used todeliver TAAs: DNA encoding TAAs can be used 1) to modify dendriticcells, 2) as ‘naked’ DNA-vaccine or 3) to construct recombinant viralvaccines. Recombinant vaccines and vaccine strategies have beendeveloped to induce and potentiate T-cell responses of a host to TAAs. Aparticular example of such a strategy is recombinant poxvirus vectors inwhich the tumor-associated antigen (TAA) is inserted as a transgene.Recombinant vaccinia vaccines and recombinant avipox(replication-defective) vaccines have been employed to stimulate immuneresponse towards the TAA; the use of diversified prime and booststrategies using different vaccines; and the insertion of multipleT-cell co-stimulatory molecules into recombinant poxvirus vectors, alongwith the TAA gene, to enhance T-cell immune response to the TAA andenhance or induce anti-tumor immunity.

The invention further provides methods of inhibiting silencing of atumor associated antigen (TAA). In one embodiment, a method includesadministering to a subject with a tumor an amount of IFN-β receptoragonist sufficient to inhibit silencing of the tumor associated antigen(TAA). In one aspect, the subject has been administered a tumorassociated antigen (TAA) prior to, substantially contemporaneously withor following IFN-β receptor agonist administration.

As used herein, the term “silencing” refers to a down-regulation of TAAexpression in tumor cells, a mechanism by which tumor cells reduceantigen expression to avoid immune detection and destruction. Thus, theterms “inhibiting silencing,” “reversing silencing,” “reducingsilencing,” and grammatical variations thereof, means that thedown-regulation of TAAs observed in tumor cells is decreased orovercome. That is, “inhibiting silencing,” means that TAA expression isincreased or TAA expression is at least stabilized to the extent thatlittle if any additional reduction in TAA expression occurs in a tumorcell.

One mechanism by which TAA silencing occurs is through suppression orinhibition of TAA gene expression at the transcriptional level, whichmay occur by what is referred to in the art as“gene silencing,” or by amechanism in which the gene promoter is inhibited (Kurnick et al. (2001)J Immunol 167:1204; Durda et al. (2003) Mol Cancer Res 1:411). “Genesilencing” is believed to occur through chromatin remodeling or proteinsthat bind DNA and that directly or indirectly inhibit transcription ofthe gene. Promoter based inhibition can also occur by positive ornegative influences on transcription factors required for genetranscription. An additional mechanism by which TAA silencing occurs isthrough increased TAA protein degradation or reduced TAA proteinstability. The invention includes “inhibiting,” “reversing” and“reducing” TAA silencing, regardless of the biological mechanism.

The invention additionally provides methods of treating a tumor. In oneembodiment, a method includes administering to a subject with a tumor anamount of IFN-β receptor agonist and tumor associated antigen (TAA)sufficient to treat the tumor. In particular aspects, the treatmentreduces tumor volume, inhibits an increase in tumor volume, stimulatestumor cell lysis or apoptosis, or reduces tumor metastasis. In anotheraspect, the subject is treated with or administered a further anti-tumortherapy (e.g., surgical resection, radiotherapy, immunotherapy orchemotherapy). In further aspects, the subject is administered anantibody or a cell that produces an antibody that specifically binds toa tumor associated antigen (TAA), an immune cell that interacts with atumor cell, or an immune-enhancing agent.

The invention moreover provides methods of treating a subject having orat risk of having a tumor. In one embodiment, a method includesadministering to a subject an amount of IFN-β receptor agonist and tumorassociated antigen (TAA) sufficient to treat the subject. In one aspect,the subject is a candidate for, is undergoing, or has undergoneanti-tumor therapy. In an additional aspect, the subject is administeredan immune cell that interacts with a tumor cell.

Methods of increasing effectiveness of an anti-tumor therapy are alsoprovided. In one embodiment, a method includes administering to asubject that is undergoing or has undergone tumor therapy, an amount ofIFN-β receptor agonist and tumor associated antigen (TAA) sufficient toincrease effectiveness of the anti-tumor therapy.

As used herein, the term “increase effectiveness,” “promoteeffectiveness,” or “improve effectiveness,” when used in reference to atherapy, such as an anti-tumor therapy or treatment protocol incombination with IFN-β receptor agonist alone or in combination withtumor associated antigen (TAA), means that the overall therapy isimproved relative to the therapy without IFN-β receptor agonist or tumorassociated antigen (TAA) treatment. Thus, the detectable or measurabletherapeutic benefit to a subject, as set forth herein, is greater withIFN-β receptor agonist or tumor associated antigen (TAA) treatment, thanin the absence of IFN-β receptor agonist or tumor associated antigen(TAA) treatment.

Non-limiting examples of IFN-β receptor agonists include, for example,IFN-β. Mammalian IFN-β sequences such as human (Gray and Goeddel (1982).Nature, 298:859); rat (Yokoyama, et al., (1997). Biochem Biophys ResCommun., 232:698); canine (Iwata, et al., (1996). J Interferon CytokineRes., 10:765); porcine (J Interferon Res., (1992). 12:153) are known inthe art. Another example of IFN agonist is anti-IFN anti-idotypicantibody (Osheroff et al. (1985). J Immunol, 135:306).

Non-limiting examples of IFN-β receptor antibodies include mammalian,human, humanized or primatized forms of heavy or light chain, V_(H) andV_(L), respectively, immunoglobulin (Ig) molecules. “Antibody” refers toany monoclonal or polyclonal immunoglobulin molecule, such as IgM, IgG,IgA, IgE, IgD, and any subclass thereof. The term “antibody” alsoincludes functional fragment of immunoglobulins, such as Fab, Fab′,(Fab)₂, Fv, Fd, scFv and sdFv, unless otherwise expressly stated.

The term “IFN-β receptor antibody” or “TAA antibody” means an antibodythat specifically binds to IFN-β receptor and a TAA antibody,respectively. Specific binding is that which is selective for an epitopepresent in IFN-β receptor or a TAA. Selective binding can bedistinguished from non-selective binding using assays known in the art(e.g., immunoprecipitation, ELISA, Western blotting).

The term “human” when used in reference to an antibody, means that theamino acid sequence of the antibody is fully human, i.e., human heavyand light chain variable and constant regions. All of the antibody aminoacids are coded for in the human DNA antibody sequences or exist in ahuman antibody. An antibody that is non-human may be made fully human bysubstituting the non-human amino acid residues with amino acid residuesthat exist in a human antibody. Amino acid residues present in humanantibodies, CDR region maps and human antibody consensus residues areknown in the art (see, e.g., Kabat, Sequences of Proteins ofImmunological Interest, 4^(th) Ed.US Department of Health and HumanServices. Public Health Service (1987); Chothia and Lesk (1987). J. Mol.Biol. 186:651; Padlan (1994). Mol. Immunol. 31:169; and Padlan (1991).Mol. Immunol. 28:489). Methods of producing human antibodies are knownin the art (see, for example, WO 02/43478 and WO 02/092812).

The term “humanized” when used in reference to an antibody, means thatthe amino acid sequence of the antibody has non-human amino acidresidues (e.g., mouse, rat, goat, rabbit, etc.) of one or moredetermining regions (CDRs) that specifically bind to the desired antigenin an acceptor human immunoglobulin molecule, and one or more humanamino acid residues in the Fv framework region (FR), which are aminoacid residues that flank the CDRs. Human framework region residues ofthe immunoglobulin can be replaced with corresponding non-humanresidues. Residues in the human framework regions can therefore besubstituted with a corresponding residue from the non-human CDR donorantibody. A humanized antibody may include residues, which are foundneither in the human antibody nor in the donor CDR or frameworksequences.

Methods of producing humanized antibodies are known in the art (see, forexample, U.S. Pat. Nos. 5,225,539; 5,530,101, 5,565,332 and 5,585,089;Riechmann, et al., (1988). Nature 332:323; EP 239,400; WO91/09967; EP592,106; EP 519,596; Padlan (1991). Molecular Immunol. 28:489; Studnickaet al., (1994). Protein Engineering 7:805; and Roguska. et al., (1994).Proc. Nat'l. Acad. Sci. USA 91:969).

Antibodies referred to as “primatized” in the art are within the meaningof “humanized” as used herein, except that the acceptor humanimmunoglobulin molecule and framework region amino acid residues may beany primate residue, in addition to any human residue.

The invention includes IFN-β peptides and mimetics, IFN-β receptoragonist peptides and mimetics, and modified (variant) forms, providedthat the modified form retains, at least partial activity or function ofunmodified or reference peptide or mimetic. For example, a modifiedIFN-β peptide or mimetic will retain at least a part of a TAA inducingactivity. Modified (variant) peptides can have one or more amino acidresidues substituted with another residue, added to the sequence ordeleted from the sequence. Specific examples include one or more aminoacid substitutions, additions or deletions (e.g., 1-3, 3-5, 5-10, 10-20,or more). A modified (variant) peptide can have a sequence with 50%,60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or more identity to areference sequence (e.g., IFN-β). The crystal structure of recombinantinterferon-beta (IFN-beta) can also be employed to predict the effect ofIFN-β modifications (Senda, et al., (1992). EMBO J. 11:3193).

As used herein, the terms “mimetic” and “mimic” refer to a syntheticchemical compound which has substantially the same structural and/orfunctional characteristics as the reference molecule. The mimetic can beentirely composed of synthetic, non-natural amino acid analogues, or canbe a chimeric molecule including one or more natural peptide amino acidsand one or more non-natural amino acid analogs. The mimetic can alsoincorporate any number of natural amino acid conservative substitutionsas long as such substitutions do not destroy activity. As withpolypeptides which are conservative variants, routine testing can beused to determine whether a mimetic has detectable TAA inducingactivity.

Peptide mimetic compositions can contain any combination of non-naturalstructural components, which are typically from three structural groups:a) residue linkage groups other than the natural amide bond (“peptidebond”) linkages; b) non-natural residues in place of naturally occurringamino acid residues; or c) residues which induce secondary structuralmimicry, i.e., induce or stabilize a secondary structure, e.g., a betaturn, gamma turn, beta sheet, alpha helix conformation, and the like.For example, a polypeptide can be characterized as a mimetic when one ormore of the residues are joined by chemical means other than an amidebond. Individual peptidomimetic residues can be joined by amide bonds,non-natural and non-amide chemical bonds other chemical bonds orcoupling means including, for example, glutaraldehyde,N-hydroxysuccinimide esters, bifunctional maleimides,N,N′-dicyclohexylcarbodiimide (DCC) or N,N′-diisopropylcarbodiimide(DIC). Linking groups alternative to the amide bond include, forexample, ketomethylene (e.g., —C(═O)—CH₂— for —C(═O)—NH—),aminomethylene (CH₂—NH), ethylene, olefin (CH═CH), ether (CH₂—O),thioether (CH₂—S), tetrazole (CN₄—), thiazole, retroamide, thioamide, orester (see, e.g., Spatola (1983) in Chemistry and Biochemistry of AminoAcids, Peptides and Proteins, Vol. 7, pp 267-357, “Peptide and BackboneModifications,” Marcel Decker, NY).

A “conservative substitution” is the replacement of one amino acid by abiologically, chemically or structurally similar residue. Biologicallysimilar means that the substitution is compatible with biologicalactivity, e.g., a TAA inducing activity. Structurally similar means thatthe amino acids have side chains with similar length, such as alanine,glycine and serine, or having similar size. Chemical similarity meansthat the residues have the same charge or are both hydrophilic orhydrophobic. Particular examples include the substitution of onehydrophobic residue, such as isoleucine, valine, leucine or methioninefor another, or the substitution of one polar residue for another, suchas the substitution of arginine for lysine, glutamic for aspartic acids,or glutamine for asparagine, serine for threonine, and the like.

A specific example of an IFN-β variant is Betaseron, an analogue ofhuman beta-interferon in which serine is substituted for cysteine atposition 17. A specific example of a IFN-β mimetic is SYR6 (Sato andSone, (2003). Biochem J., 371(Pt 2):603). Modified IFN-β sequencecandidates for use in the invention are described, for example, in U.S.Pat. Nos. 6,514,729—recombinant interferon-beta muteins;4,793,995—modified (1-56) beta interferons; 4,753,795—modified (80-113)beta interferons; and 4,738,845—modified (115-145) beta interferons.

Peptides and peptidomimetics can be produced and isolated using anymethod known in the art. Peptides can be synthesized, whole or in part,using chemical methods known in the art (see, e.g., Caruthers (1980).Nucleic Acids Res. Symp. Ser. 215; Horn (1980). Nucleic Acids Res. Symp.Ser. 225; and Banga, A. K., Therapeutic Peptides and Proteins,Formulation, Processing and Delivery Systems (1995) Technomic PublishingCo., Lancaster, Pa.). Peptide synthesis can be performed using varioussolid-phase techniques (see, e.g., Roberge (1995) Science 269:202;Merrifield (1997). Methods Enzymol. 289:3) and automated synthesis maybe achieved, e.g., using the ABI 431A Peptide Synthesizer (Perkin Elmer)in accordance with the manufacturer's instructions.

Individual synthetic residues and polypeptides incorporating mimeticscan be synthesized using a variety of procedures and methodologies knownin the art (see, e.g., Organic Syntheses Collective Volumes, Gilman, etal. (Eds) John Wiley & Sons, Inc., NY). Peptides and peptide mimeticscan also be synthesized using combinatorial methodologies. Techniquesfor generating peptide and peptidomimetic libraries are well known, andinclude, for example, multipin, tea bag, and split-couple-mix techniques(see, for example, al-Obeidi (1998). Mol. Biotechnol. 9:205; Hruby(1997). Curr. Opin. Chem. Biol. 1:114; Ostergaard (1997). Mol. Divers.3:17; and Ostresh (1996). Methods Enzymol. 267:220). Modified peptidescan be further produced by chemical modification methods (see, forexample, Belousov (1997). Nucleic Acids Res. 25:3440; Frenkel (1995).Free Radic. Biol. Med. 19:373; and Blommers (1994). Biochemistry33:7886).

Peptides can also be synthesized and expressed as fusion proteins withone or more additional domains linked thereto for producing a moreimmunogenic peptide, or to more readily isolate a recombinantlysynthesized peptide. Domains facilitating isolation include, forexample, metal chelating peptides such as polyhistidine tracts andhistidine-tryptophan modules that allow purification on immobilizedmetals; protein A domains that allow purification on immobilizedimmunoglobulin; and the domain utilized in the FLAGS extension/affinitypurification system (Immunex Corp, Seattle Wash.). The inclusion of acleavable linker sequence such as Factor Xa or enterokinase (Invitrogen,San Diego Calif.) between a purification domain and the peptide can beused to facilitate peptide purification. For example, an expressionvector can include a peptide-encoding nucleic acid sequence linked tosix histidine residues followed by a thioredoxin and an enterokinasecleavage site (see e.g., Williams (1995). Biochemistry 34:1787; andDobeli (1998). Protein Expr. Purif. 12:404). The histidine residuesfacilitate detection and purification of the fusion protein while theenterokinase cleavage site provides a means for purifying the peptidefrom the remainder of the fusion protein. Technology pertaining tovectors encoding fusion proteins and application of fusion proteins isknown in the art (see e.g., Kroll (1993). DNA Cell. Biol., 12:441).

The invention includes any metastatic or non-metastatic tumor, cancer,malignancy or neoplasia of any cell or tissue origin. The tumor may bein any stage, e.g., a stage I, II, III, IV or V tumor, or in remission.

As used herein, the terms “tumor,” “cancer,” “malignancy,” and“neoplasia” are used interchangeably and refer to a cell or populationof cells whose growth, proliferation or survival is greater than growth,proliferation or survival of a normal counterpart cell, e.g. a cellproliferative or differentiative disorder. Such disorders can affectvirtually any cell or tissue type, e.g., carcinoma, sarcoma, melanoma,neural, and reticuloendothelial or haematopoietic neoplastic disorders(e.g., myeloma, lymphoma or leukemia). A tumor can arise from amultitude of primary tumor types, including but not limited to breast,lung, thyroid, head and neck, brain, lymphoid, gastrointestinal (mouth,esophagus, stomach, small intestine, colon, rectum), genito-urinarytract (uterus, ovary, cervix, bladder, testicle, penis, prostate),kidney, pancreas, liver, bone, muscle, skin, and metastasize to othersecondary sites.

Cells comprising a tumor may be aggregated in a cell mass or bedispersed. A “solid tumor” refers to neoplasia or metastasis thattypically aggregates together and forms a mass. Specific examplesinclude visceral tumors such as melanomas, breast, pancreatic, uterineand ovarian cancers, testicular cancer, including seminomas, gastric orcolon cancer, hepatomas, adrenal, renal and bladder carcinomas, lung,head and neck cancers and brain tumors/cancers.

Carcinomas refer to malignancies of epithelial or endocrine tissue, andinclude respiratory system carcinomas, gastrointestinal systemcarcinomas, genitourinary system carcinomas, testicular carcinomas,breast carcinomas, prostatic carcinomas, endocrine system carcinomas,and melanomas. Melanoma refers to malignant tumors of melanocytes andother cells derived from pigment cell origin that may arise in the skin,the eye (including retina), or other regions of the body, including thecells derived from the neural crest that also gives rise to themelanocyte lineage. A pre-malignant form of melanoma, known asdysplastic nevus or dysplastic nevus syndrome, is associated withmelanoma development.

Exemplary carcinomas include those forming from the uterine cervix,lung, prostate, breast, head and neck, colon, pancreas, testes, adrenal,kidney, esophagus, stomach, liver and ovary. The term also includescarcinosarcomas, e.g., which include malignant tumors composed ofcarcinomatous and sarcomatous tissues. Adenocarcinoma includes acarcinoma of a glandular tissue, or in which the tumor forms a glandlike structure.

Sarcomas refer to malignant tumors of mesenchymal cell origin. Exemplarysarcomas include for example, lymphosarcoma, liposarcoma, osteosarcoma,chondrosarcoma, leiomyosarcoma, rhabdomyosarcoma and fibrosarcoma.

Neural neoplasias include glioma, glioblastoma, meningioma,neuroblastoma, retinoblastoma, astrocytoma, oligodendrocytoma

A “liquid tumor” refers to neoplasia of the reticuloendothelial orhaematopoetic system, such as a lymphoma, myeloma and leukemia, orneoplasia that is diffuse in nature, as they do not typically form asolid mass. Particular examples of leukemias include acute and chroniclymphoblastic, myeolblastic and multiple myeloma. Typically, suchdiseases arise from poorly differentiated acute leukemias, e.g.,erythroblastic leukemia and acute megakaryoblastic leukemia. Specificmyeloid disorders include, but are not limited to, acute promyeloidleukemia (APML), acute myelogenous leukemia (AML) and chronicmyelogenous leukemia (CML); lymphoid malignancies include, but are notlimited to, acute lymphoblastic leukemia (ALL), which includes B-lineageALL and T-lineage ALL, chronic lymphocytic leukemia (CLL),prolymphocytic leukemia (PLL), hairy cell leukemia (HLL) andWaldenstrom's macroglobulinemia (WM). Specific malignant lymphomasinclude, non-Hodgkin lymphoma and variants, peripheral T cell lymphomas,adult T cell leukemia/lymphoma (ATL), cutaneous T-cell lymphoma (CTCL),large granular lymphocytic leukemia (LGF), Hodgkin's disease andReed-Sternberg disease.

As used herein, an “anti-tumor,” “anti-cancer” or “anti-neoplastic”treatment, therapy, activity or effect means any compound, agent,therapy or treatment regimen or protocol that inhibits, decreases,retards, slows, reduces or prevents tumor, cancer or neoplastic growth,metastasis, proliferation or survival, in vitro or in vivo. Particularnon-limiting examples of anti-tumor therapy include chemotherapy,immunotherapy, radiotherapy (ionizing or chemical), local thermal(hyperthermia) therapy and surgical resection. Any compound, agent,therapy or treatment regimen or protocol having an anti-cellproliferative activity or effect can be used in combination with anIFN-β receptor agonist, or a compound or agent having IFN-β activity inaccordance with the invention.

Anti-proliferative or anti-tumor compounds, agents, therapies ortreatments can operate by biological mechanisms that disrupt, interrupt,inhibit or delay cell cycle progression or cell proliferation; stimulateor enhance apoptosis or cell death, inhibit nucleic acid or proteinsynthesis or metabolism, inhibit cell division, or decrease, reduce orinhibit cell survival, or production or utilization of a necessary cellsurvival factor, growth factor or signaling pathway (extracellular orintracellular). Non-limiting examples of chemical agent classes havinganti-cell proliferative and anti-tumor activities include alkylatingagents, anti-metabolites, plant extracts, plant alkaloids, nitrosoureas,hormones, nucleoside and nucleotide analogues. Specific examples ofdrugs include cyclophosphamide, azathioprine, cyclosporin A,prednisolone, melphalan, chlorambucil, mechlorethamine, busulphan,methotrexate, 6-mercaptopurine, thioguanine, 5-fluorouracil, cytosinearabinoside, AZT, 5-azacytidine (5-AZC) and 5-azacytidine relatedcompounds such as decitabine (5-aza-2′ deoxycytidine), cytarabine,1-beta-D-arabinofuranosyl-5-azacytosine and dihydro-5-azacytidine(Goffin et al. (2002). Ann Oncol. 13:1699; Gaubert (2000). Eur J Med.Chem. 35:1011), bleomycin, actinomycin D, mithramycin, mitomycin C,carmustine, lomustine, semustine, streptozotocin, hydroxyurea,cisplatin, mitotane, procarbazine, dacarbazine, taxol, vinblastine,vincristine, doxorubicin and dibromomannitol.

Additional chemotherapeutic and biotherapeutic agents are known in theart and can be employed. For example, monoclonal antibodies that bindtumor cells or oncogene products, such as Rituxan® and Herceptin(Trastuzumab)(anti-Her-2 neu antibody), Bevacizumab (Avastin), Zevalin,Bexxar, Oncolym, 17-1A(Edrecolomab), 3F8 (anti-neuroblastoma antibody),MDX-CTLA4, Campath®, Mylotarg, IMC-C225 (Cetuximab), aurinstatinconjugates of cBR96 and cAC10 (Doronina et al. (2003). Nat Biotechnol21:778) can be used in combination with an IFN-β receptor agonist, or acompound or agent having IFN-β activity in accordance with theinvention.

Compounds or agents having similar activity as IFN-β (a TAA-inducingactivity) may or may not act through IFN-β receptor. For example, TAAregulatory regions are likely to include one or more genetic regulatoryelements such that TAA expression is responsive to other inducers andsuppressor molecules (i.e., other than IFN-β or IFN-β agonists). Thus,the invention may be practiced with compounds or agents that induce orsuppress expression of a TAA via one or more genetic regulatory elements(i.e., any cis-acting nucleic acid element that can directly orindirectly alter expression of a TAA).

One example of such a molecule is nuclear factor of activated T-cells(also referred to as NFAT-motif binding protein, e.g., NFATc1, c2 c3 andc4), which is a family of transcription factors that participate inmediating signal transduction. Modulating (increasing or decreasing) anactivity or function of an NFAT-motif binding protein is likely tomodulate TAA expression. As used herein, the terms “activity” or“function” when used to modify “NFAT-motif binding protein,” means thatNFAT-motif binding protein is altered so as to alter TAA expression. Forexample, increased or decreased binding of an NFAT binding protein to aTAA regulatory region is one mechanism by which an NFAT-motif bindingprotein could regulate TAA expression.

Thus, the invention includes methods of modulating TAA expression,increasing an immune response against a tumor cell, increasingeffectiveness of an anti-tumor therapy, treating a subject having or atrisk of having a tumor, treating a tumor and inhibiting silencing of atumor associated antigen (TAA), with an agent or compound that modulatesan activity or function of an NFAT-motif binding protein. In respectiveembodiments, a method includes contacting a cell capable of expressing aTAA with a compound that modulates an activity of an NFAT-motif bindingprotein in an amount sufficient to increase expression of a tumorassociated antigen (TAA) of the cell; increase an immune responseagainst the tumor cell; increase effectiveness of the anti-tumortherapy; treat the subject, treat the tumor; and inhibit silencing of atumor associated antigen (TAA).

Specific non-limiting examples of compounds that modulate an activity ofan NFAT-motif binding protein include a calcium flux modulator (e.g.,ionomycin or verapimil), VIVIT (Pu, et al. (2003). Circ Res 92:725),gossypol (Baumgrass, et al. (2001). J Biol Chem 276:47914),N-substituted benzamides (Lindgren, et al. (2001). Mol Immunol 38:267),rapamycin (Marx, et al. (1995). Circ Res 76:412),quinazoline-2,4-diones, 1-3, and pyrrolo[3,4-d]pyrimidine-2,4-diones,4-8 (Michne, et al. (1995). J Med Chem 38:2557),1alpha,25-dihydroxyvitamin D3 (Takeuchi, et al. (1998). J Immunol160:209), FK506 (Rovira, et al. (2000). Curr Med Chem 7:673), FK520(Marx, et al. (1995). Circ Res 76:412), cyclosporin (Rovira, et al.(2000). Curr Med Chem 7:673), 3,5-Bis(trifluoromethyl)pyrazoles (Djuric,et al. (2000). J Med Chem 43:2975), dithiocarbamates (Martinez-Martinez,et al. (1997). Mol Cell Biol 17:6437), Vasoactive intestinal peptide(VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP)(Ganea and Delgado (2002). Crit. Rev Oral Biol Med 13:229),carboxyamidotriazole (Faehling et al. (2002). Faseb J 16:1805), morphine(Wang, et al. (2003). J Biol Chem Jul 3 [Epub ahead of print]),C32-O-arylethyl ether derivatives of ascomycin (Armstrong, et al.(1999). Bioorg Med Chem Lett 9:2089), Ascomycin macrolactam derivativeSDZ ASM 981 (Hultsch, et al. (1998). Arch Dermatol Res 290:501), andMCIP1 (Vega, et al. (2002). J Biol Chem 277:30401).

Additional examples of compounds that modulate an activity of anNFAT-motif binding protein include an NFAT antisense nucleic acid orRNAi, NFAT binding protein (e.g., an antibody; see, for example, Lyakhet al., Mol Cell Biol. (1997). 17:2475) or dominant negative NFATpolypeptide (see, for example, Schubert et al. (2003). J Cell Biol161:861; van Rooij et al. (2002). J Biol Chem 277:48617).

Antisense can be designed based on NFAT nucleic acid sequences availablein the database. Antisense includes single, double or triple strandedpolynucleotides and peptide nucleic acids (PNAs) that bind RNAtranscript or DNA. For example, a single stranded nucleic acid cantarget NFAT binding protein transcript (e.g., mRNA). Oligonucleotidesderived from the transcription initiation site of the gene, e.g.,between positions −10 and +10 from the start site, are a particular oneexample. Triplex forming antisense can bind to double strand DNA therebyinhibiting transcription of the gene. The use of double stranded RNAsequences (known as “RNAi”) for inhibiting gene expression is known inthe art (see, e.g., Kennerdell et al., (1998). Cell 95:1017; Fire etal., (1998). Nature, 391:806). Double stranded RNA sequences from anNFAT binding protein coding region may therefore be used to inhibitexpression.

Compounds and agents having IFN-β activity (including IFN-β receptoragonists) may be more or less potent than IFN-β. Thus, a compound canhave significantly less (e.g., 10% of the potency or activity) or more(e.g., 150-500%, or greater, potency or activity) of IFN-β.

Compounds or agents having IFN-β activity (e.g., increase or induceexpression of a tumor associated antigen) may be used alone or incombination with IFN-β, IFN-β receptor agonist, or other compounds,agents, treatment or therapies having an anti-tumor effect or activity.For example, administering one or more TAA's expressed by a tumor incombination with the compound or agent having IFN-β activity canincrease immune response towards a tumor that expresses or is induced toexpress the TAA, thereby increasing the effectiveness of the anti-tumortherapy.

In an invention method of administering one or more TAAs with IFN-β, anIFN-β receptor agonist, or a compound or agent having TAA-inducingactivity as IFN-β, the two components need not be administeredsubstantially contemporaneously with each other. In other words, a TAAmay be administered to a subject within one or more hours (e.g., 1-3,3-6, 6-12, 12-24, 24-48, 24-72 hours), days (e.g., 1-3, 3-5, 5-7,7-10,10-14 days, 14-30 days) or months (1-6) before or after IFN-β an IFN-βreceptor agonist, or a compound or agent having TAA-inducing activity asIFN-β, administration. Accordingly, one or more TAAs can be administeredprior to, substantially contemporaneous with or following administrationof IFN-β, an IFN-β receptor agonist, or a compound or agent havingsimilar activity as IFN-β in any order desired.

If a subject is first administered TAA (singly or multiple times), thesubject may subsequently be administered IFN-β, an IFN-β receptoragonist, or a compound or agent having TAA-inducing activity as IFN-β,multiple times. Likewise, if a subject is first administered IFN-β, anIFN-β receptor agonist, or a compound or agent having TAA-inducingactivity as IFN-β singly or multiple times, the subject may besubsequently administered TAA multiple times.

A subject may be first administered a TAA, and subsequently administeredIFN-β, an IFN-β receptor agonist, or a compound or agent havingTAA-inducing activity as IFN-β. Alternatively, a subject may be firstadministered IFN-β, an IFN-β receptor agonist, or a compound or agenthaving TAA-inducing activity as IFN-β, and subsequently administered aTAA. A subject may also be given multiple administrations of TAA andIFN-β, an IFN-β receptor agonist, or a compound or agent havingTAA-inducing activity as IFN-β, in any sequence.

Any compound, agent, therapy or treatment having an immune-stimulatingor enhancing activity or effect can be used in combination with an IFN-βreceptor agonist, or a compound or agent having TAA-inducing activity asIFN-β, in accordance with the invention. As used herein, the term“immune enhancing,” when used in reference to such a compound, agent,therapy or treatment, means that the compound provides an increase,stimulation, induction or promotion of an immune response, humoral orcell-mediated. Such therapies can enhance immune response generally, orenhance immune response to the specific tumor. Specific non-limitingexamples of immune enhancing agents include monoclonal, polyclonalantibody and mixtures thereof (e.g., that specifically bind to a TAA).

Immune cells that interact with a tumor cell include lymphocytes, plasmacells, B-cells expressing antibody against TAA, NK cells, LAK cells andmacrophages. Immune cells include cells that enhance or stimulate animmune response against TAA (e.g., dendritic cells or antigen presentingcells) are considered “immune enhancing”. In addition, a mammalian ornon-mammalian cell that expresses an antibody (e.g., plasma cell, B-cellor a mammalian or non-mammalian cell transfected with a nucleic acidencoding the antibody) that specifically binds to a TAA, can be used inaccordance with the invention. An immune cell that targets a tumor cellcan be used in accordance with the invention. For example, adoptiveimmunotherapy, in which tumor-infiltrating or peripheral bloodlymphocytes can be infused into a tumor patient, following optionalstimulation with a cytokine.

Immune stimulating molecules (Dredge et al. (2002) Cancer Immunol

Immunother 51:521), such as Flt3 ligand (Disis et al. (2002) Blood99:2845) and cytokines (e.g., cell growth, proliferation, chemotacticand survival factors) that enhance or stimulate immunogenicity of TAAare considered “immune enhancing,” and can be administered prior to,substantially contemporaneously with or following administration ofIFN-β receptor agonist, or a compound or agent having TAA-inducingactivity as IFN-(Nohria et al. (1994). Biotherapy 7:261; Pardoll (1995).Annu Rev Immunol 13:399; and Ahlers et al. (1997) J Immunol 158:3947).Specific non-limiting examples of cytokines include IL-2, IL-1α, IL-1β,IL-3, IL-7, granulocyte-macrophage-colony stimulating factor (GMCSF),IFN-γ, IL-12, and TNF-α (Riker et al. (1999). Surgery 126:112;Scheibenbogen et al. (2002). Int J Cancer 98:409; Disis et al. (2002).Blood 99:2845; Schiller et al. (1990). J Clin Invest 86:1211; Chen etal. (2001). Gene Ther 8:316; Elzey et al. (2001). Int J Cancer 94:842).GMCSF stimulates antigen-presenting cells and exhibits anti-tumoractivity, including against leukemia, melanoma, breast carcinoma,prostate carcinoma and renal cell carcinoma, can be used in accordancewith the invention.

Molecules that that down-regulate the effects of TH1 immune responseinhibitors are also considered as “immune enhancing.” Specificnon-limiting examples include antibodies to IL-10 or IL-10 receptor(Murray et al. (2003) Infect Dis 188:458), IL-4 and IL-5, therebyup-regulating the TH1 immune response

Kinase inhibitors that enhance or stimulate TAA expression includeGleevec (STI571) and inhibitors of protein kinases (e.g. AKT inhibitor,H-89, PD98059, PD184352, U0126, HA1077, forskolin and Y27632). Suchkinase inhibitors may synergize with other compounds (e.g., IFN-β thatstimulate, enhance or increase TAA expression.

“Gene silencing inhibitors” including DNA methyl transferase inhibitorssuch as 5-azacytosine and inhibitors of histone deacetylase such astrichostatin A are considered as “immune enhancing.” IFN-β may alsosynergize with such inhibitors.

Adjuvants refer to a class of substances which when added to an antigenimprove the immune response. Examples include compounds which promoteuptake by accessory cells (e.g. macrophages and dendritic cells) whichprocess antigen, such as alum (aluminum hydroxide), incomplete Freund'sadjuvant, complete Freund's adjuvant, Ribi, Montanide ISA™51, GERBUvaccine adjuvant, CAP vaccine adjuvant, SLN (solid lipid nanoparticles),CpG DNA and RC529 adjuvant.

The invention therefore also provides methods of treating a tumor,methods of treating a subject having or at risk of having a tumor, andmethods of increasing effectiveness of an anti-tumor therapy. Inrespective embodiments, a method includes administering to a subjectwith a tumor an amount of IFN-β receptor agonist and an antibody or acell that produces an antibody that specifically binds to a tumorassociated antigen (TAA) sufficient to treat the tumor; administering tothe subject an amount of IFN-β receptor agonist and an antibody or acell that produces an antibody that specifically binds to a tumorassociated antigen (TAA) sufficient to treat the subject; andadministering to a subject that is undergoing or has undergone tumortherapy, an amount of IFN-β receptor agonist and an antibody or a cellthat produces an antibody that specifically binds to a tumor associatedantigen (TAA) sufficient to increase effectiveness of the anti-tumortherapy. In various aspects, the cell producing an antibody thatspecifically binds to a tumor associated antigen (TAA) is selected froma plasma cell, B-cell, or a mammalian or non-mammalian cell transfectedwith a nucleic acid encoding the antibody.

The invention therefore further provides methods of treating a tumor,methods of treating a subject having or at risk of having a tumor, andmethods of increasing effectiveness of an anti-tumor therapy. Inrespective embodiments, a method includes administering to a subjectwith a tumor an amount of IFN-β receptor agonist and an immune cell thatinteracts with a tumor cell sufficient to treat the tumor; administeringto the subject an amount of IFN-β receptor agonist and an immune cellthat interacts with a tumor cell sufficient to treat the subject; andadministering to a subject that is undergoing or has undergone tumortherapy, an amount of IFN-β receptor agonist and an immune cell thatinteracts with a tumor cell sufficient to increase effectiveness of theanti-tumor therapy. In various aspects, the cell is selected from a Tcell, NK cell, LAK cell, monocyte or macrophage. In an additionalaspect, the cell has been pre-selected to bind to an antigen (e.g., aTAA) expressed by the tumor (e.g., T lymphocytes selected for strongavidity to TAA as presented on HLA molecules, Dudley et al. (2002).Science 298:850; Yee et al. (2002). PNAS 99:16168).

Methods of the invention include providing a detectable or measurabletherapeutic benefit to a subject. A therapeutic benefit is any objectiveor subjective transient or temporary, or longer term improvement in thecondition. Thus, a satisfactory clinical endpoint is achieved when thereis an incremental improvement in the subjects condition or a partialreduction in the severity or duration of one or more associated adversesymptoms or complications or inhibition or reversal of one or more ofthe physiological, biochemical or cellular manifestations orcharacteristics of the disease. A therapeutic benefit or improvement(“ameliorate” is used synonymously) therefore need not be completeablation of the tumor or any or all adverse symptoms or complicationsassociated with the tumor. For example, inhibiting an increase in tumorcell mass (stabilization of a disease) can increase the subjectslifespan (reduce mortality) even if only for a few days, weeks ormonths, even though complete ablation of the tumor has not resulted.

Particular examples of therapeutic benefit or improvement include areduction in tumor volume (size or cell mass), inhibiting an increase intumor volume, a slowing or inhibition of tumor worsening or progression,stimulating tumor cell lysis or apoptosis, reducing or inhibiting tumormetastasis, reduced mortality, prolonging lifespan. Adverse symptoms andcomplications associated with tumor, neoplasia, and cancer that can bereduced or decreased include, for example, nausea, lack of appetite, andlethargy. Thus, a reduction in the severity or frequency of symptoms, animprovement in the subjects subjective feeling, such as increasedenergy, appetite, psychological well being, are examples of therapeuticbenefit

The doses or “sufficient amount” for treatment to achieve a therapeuticbenefit or improvement are effective to ameliorate one, several or alladverse symptoms or complications of the condition, to a measurableextent, although reducing or inhibiting a progression or worsening ofthe condition or an adverse symptom, is a satisfactory outcome. The dosemay be proportionally increased or reduced as indicated by the status ofthe disease being treated or the side effects of the treatment. Dosesalso considered sufficient are those that result in a reduction of theuse of another therapeutic regimen or protocol. For example, an IFN-βreceptor agonist and one or more TAAs is considered as having atherapeutic effect if administration results in less chemotherapeuticdrug, radiation or immunotherapy being required for tumor treatment.

As is typical for treatment protocols, some subjects will exhibitgreater or less response to treatment. Thus, appropriate amounts willdepend upon the condition treated (e.g., the type or stage of thetumor), the therapeutic effect desired, as well as the individualsubject (e.g., the bioavailability within the subject, gender, age,etc.).

Subjects appropriate for treatment include those having or at risk ofhaving a tumor cell, those undergoing as well as those who areundergoing or have undergone anti-tumor therapy, including subjectswhere the tumor is in remission. The invention is therefore applicableto treating a subject who is at risk of a tumor or a complicationassociated with a tumor. Prophylactic methods are therefore included.

Subjects include those who have risk factors associated with tumordevelopment. For example, subjects at risk for developing melanomainclude fair skin, high numbers of naevi (dysplastic nevus), sunexposure (ultraviolet radiation), patient phenotype, family history, andhistory of a previous melanoma. Subjects at risk for developing cancercan be identified with genetic screens for tumor associated genes, genedeletions or gene mutations. Subjects at risk for developing breastcancer lack Brcal, for example. Subjects at risk for developing coloncancer have deleted or mutated tumor suppressor genes, such asadenomatous polyposis coli (APC), for example.

The term “subject” refers to animals, typically mammalian animals, suchas a non human primate (apes, gibbons, chimpanzees, orangutans,macaques), a domestic animal (dogs and cats), a farm animal (horses,cows, goats, sheep, pigs), experimental animal (mouse, rat, rabbit,guinea pig) and humans. Subjects include animal disease models, forexample, a rodent model for testing in vivo efficacy of IFN-β receptoragonist and one or more TAAs (e.g., a tumor animal model).

IFN-

receptor agonist, compounds and agents having a TAA-inducing activity asIFN-β can be administered in a conventional dosage form prepared bycombining IFN-β receptor agonist, or a compound or agent havingTAA-inducing activity as IFN-β with a conventional pharmaceuticallyacceptable carrier or diluent according to known techniques. Thepharmaceutically acceptable carrier or diluent is dictated by the amountof active ingredient with which it is to be combined, the route ofadministration and other known variables.

Pharmaceutical compositions include “pharmaceutically acceptable” and“physiologically acceptable” carriers, diluents or excipients. As usedherein, the term “pharmaceutically acceptable” and “physiologicallyacceptable,” when referring to carriers, diluents or excipients includessolvents (aqueous or non-aqueous), detergents, solutions, emulsions,dispersion media, coatings, isotonic and absorption promoting ordelaying agents, compatible with pharmaceutical administration and withthe other components of the formulation. Such formulations can becontained in a tablet (coated or uncoated), capsule (hard or soft),microbead, emulsion, powder, granule, crystal, suspension, syrup orelixir.

Pharmaceutical compositions can be formulated to be compatible with aparticular route of administration. Compositions for parenteral,intradermal, or subcutaneous administration can include a sterilediluent, such as water, saline solution, fixed oils, polyethyleneglycols, glycerine, propylene glycol or other synthetic solvents. Thepreparation may contain one or more preservatives to preventmicroorganism growth (e.g., antibacterial agents such as benzyl alcoholor methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose).

Pharmaceutical compositions for injection include sterile aqueoussolutions (where water soluble) or dispersions and sterile powders forthe extemporaneous preparation of sterile injectable solutions ordispersion. For intravenous administration, suitable carriers includephysiological saline, bacteriostatic water, Cremophor EL™ (BASF,Parsippany, N.J.) or phosphate buffered saline (PBS). The carrier can bea solvent or dispersion medium containing, for example, water, ethanol,polyol (e.g., glycerol, propylene glycol, and polyetheylene glycol), andsuitable mixtures thereof. Fluidity can be maintained, for example, bythe use of a coating such as lecithin, or by the use of surfactants.Antibacterial and antifungal agents include, for example, parabens,chlorobutanol, phenol, ascorbic acid and thimerosal. Including an agentthat delays absorption, for example, aluminum monostearate and gelatincan prolonged absorption of injectable compositions.

For transmucosal or transdermal administration, penetrants appropriateto the barrier to be permeated are used in the formulation. Suchpenetrants are known in the art, and include, for example, fortransmucosal administration, detergents, bile salts, and fusidic acidderivatives; for transdermal administration, ointments, salves, gels, orcreams.

Additional pharmaceutical formulations and delivery systems are known inthe art and are applicable in the methods of the invention (see, e.g.,Remington's Pharmaceutical Sciences (1990) 18th ed., Mack PublishingCo., Easton, Pa.; The Merck Index (1996) 12th ed., Merck PublishingGroup, Whitehouse, N.J.; Pharmaceutical Principles of Solid DosageForms, Technonic Publishing Co., Inc., Lancaster, Pa., (1993); andPoznansky, et al., Drug Delivery Systems, R. L. Juliano, ed., Oxford,N.Y. (1980), pp. 253-315)

Methods of identifying an agent that increases expression of a melanomatumor associated antigen (TAA) are also provided. In one embodiment, amethod includes contacting a cell capable of expressing a melanoma TAAwith a test agent (e.g., a melanoma cell); measuring the amount of TAAexpressed in the presence of the test agent; and determining whether theamount of TAA expressed is greater in the presence than in the absenceof the test agent, wherein increased TAA expression identifies the testagent as an agent that increases expression of a melanoma TAA. In oneaspect, the TAA is a differentiation antigen, e.g., Melan-A/MART-1,tyrosinase, gp100/pmel 17, TRP-1, TRP-2 or MITF-M, or an antigenicfragment thereof.

Kits that include one or more of IFN-β and IFN-β receptor agonist, or acompound or agent having a TAA-inducing activity as IFN-β packaged intosuitable packaging material, are also provided. A kit typically includesa label or packaging insert including a description of the components orinstructions for use in vitro, in vivo, or ex vivo, of the componentstherein. A kit can contain a collection of such components, e.g., IFN-βan IFN-β receptor agonist, or a compound or agent having a TAA-inducingactivity as IFN-β, and one or more TAAs.

In one embodiment, a kit includes IFN-β, an IFN-β receptor agonist, or acompound or agent having a TAA-inducing activity as IFN-β, andinstructions for treating (prophylaxis or therapeutic), a tumor of asubject. In another embodiment, the container includes one or more TAAs.In yet another embodiment, the kit or container includes an anti-tumoragent (e.g., a drug or antibody, such as an anti-TAA antibody).

The term “packaging material” refers to a physical structure housing thecomponents of the kit. The packaging material can maintain thecomponents sterilely, and can be made of material commonly used for suchpurposes (e.g., paper, corrugated fiber, glass, plastic, foil, ampules,etc.). The label or packaging insert can include appropriate writteninstructions.

Kits of the invention therefore can additionally include labels orinstructions for using the kit components in a method of the invention.Instructions can include instructions for practicing any of the methodsof the invention described herein including treatment methods. Thus, forexample, a kit can include IFN-β and one or more TAAs, together withinstructions for administering to a subject in a treatment method of theinvention.

The instructions may be on “printed matter,” e.g., on paper or cardboardwithin or affixed to the kit, or on a label affixed to the kit orpackaging material, or attached to a vial or tube containing a componentof the kit. Instructions may additionally be included on a computerreadable medium, such as a disk (floppy diskette or hard disk), opticalCD such as CD- or DVD-ROM/RAM, magnetic tape, electrical storage mediasuch as RAM and ROM and hybrids of these such as magnetic/opticalstorage media.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described herein.

All publications, patents and other references cited herein areincorporated by reference in their entirety. In case of conflict, thespecification, including definitions, controls.

As used herein, the singular forms “a”, “and,” and “the” include pluralreferents unless the context clearly indicates otherwise. Thus, forexample, reference to an “IFN-beta agonist” includes a plurality ofIFN-beta agonists and reference to “a tumor associated antigen” includesreference to one or more tumor associated antigens.

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, the following examples are intended to illustrate but notlimit the scope of invention described in the claims.

EXAMPLES Example I

This example describes exemplary materials, methods and procedures.

Cell Lines: All cell lines have been previously described. Melanomatumor cells lines, MU, MU-X, EW, were established at the MassachusettsGeneral Hospital (Ramirez-Montagut, et al. (2000). Clin. Exp. Immunol.119:11). A375 was purchased from American Type Culture Collection,(Manassas, Va.). IGR-39D, 453A and 136.2 were provided by Dr. PeterSchrier, Leiden University, Leiden, The Netherlands. MM96L was providedby Dr. P. G. Parsons, Queensland Institute of Medical Research, Herston,Australia; (+) and (−) varieties (i.e. high and low expressors ofMelan-A/MART-1 antigen) were derived by Dr. James Kurnick. The U937myelomonocytic cell line was isolated by Dr. Kenneth Nilsson, UppsalaUniversity, Uppsala, Sweden. U2-OS, a human osteosarcoma cell line asdescribed by Nelissen (Nelissen et al. (2000). Exp Hematol 28:422.).

Reagents: Antibodies against Melan-A/MART-1 (clone A103) (Chen, et al.(1996) Proc Natl Acad Sci USA 93:5915) were purchased from VectorLaboratories/NoivoCastra Laboratories (Burlingame, Calif.). Anti-gp100(clone HMB45) antibodies were obtained from Lab Vision Corp. (Fremont,Calif.). Recombinant human oncostatin M (rhOSM) was obtained from R&DSystems, (Minneapolis Minn.). Chemicals and other reagents wereAnalytical Grade and obtained from Sigma-Aldrich, (St. Louis, Mo.).Recombinant human beta-interferon-1a (Avonex®) and interferon-1b(Betaseron ®) were products of Biogen (Cambridge, Mass.) and BerlexLaboratories Inc. (Montville, N.J.), respectively.

Conditioned Medium: Conditioned medium from Melan-A/MART-1 deficientmelanoma tumor cell lines was generated by culturing cells at a startingconcentration of 5×10⁵ cells/ml in DMEM medium supplemented with between1 and 10% FBS. Supernatants were collected after 72 hours bycentrifugation of the cell cultures and filtration of the medium througha 0.2 micron filter (Millipore, Bedford, Mass.). Conditioned mediumcontaining 1% FBS was concentrated between 10 and 20 fold by collectingthe retentate from a nominal 301d) YM membrane (Centriprep, Millipore,Bedford, Mass.). In addition to tumor cell lines MU-X, EW and IGR39D,three non-melanoma cell lines were also used to generate conditionedmedium under similar conditions. These human tumor cell lines were:Daudi (B cell lymphoma); Jurkat (T cell lymphoma; MCF-7 (breastcarcinoma), which were obtained from the ATCC (American Type CultureCollection, Bethesda, Md.).

Determination of Protein Antigen in Tumor Cells via Flow CytometricAnalysis: To evaluate expression of cytoplasmic Melan-A/MART-1 antigenin melanoma tumor cell lines, cells were first fixed for 10′ in 1%paraformaldehyde; the cells are pelleted and incubated for 5′ in 0.1%saponin prior to washing and addition of monoclonal antibody specificfor Melan-A/MART-1, A-103 (Ramirez-Montagut, et al., supra) for 45′ at22° C. Following two washes, the cells were stained for 30′ withFITC-conjugated goat-anti-mouse Ig antibody (DAKO, Carpenteria, Calif.)prior to fixation in 1% paraformaldehyde and analysis by flow cytometry(FACScan, Becton-Dickinson, Mt. View, Calif.). Histograms offluorescence staining were generated for comparison ofanti-Melan-A/MART-1 staining of various cell populations. Mean channelfluorescence was calculated using the “LYSIS” software provided by themanufacturer. Gp100 expression was determined similarly using the HMB45monoclonal antibody.

Cytotoxicity Assays: TIL were assayed for the ability to lyse melanomatarget cells in 4 hours via a ⁵¹Cr-release assay, as previouslydescribed (Ramirez-Montagut, et al., supra). The melanoma target cellswith high constitutive expression of Melan-A/MART-1 were generated bylow density culture (1-2×10⁵/ml). These Melan-A/MART-1 expressing cellswere compared with respect to their susceptibility to cytolysis to thesame cells cultured for 3 to 6 days in the presence of conditionedmedium from the Melan-A/MART-1 negative variant, MU-X, to derive targetcells with low Melan-A/MART-1 expression. Low Melan-A/MART-1 expressingcells were further assayed after pulsing with Melan-A/MART-1 peptideamino acids 27-35 (AAGIGILTV; SEQ ID NO:1); (Zhai, et al. (1996). J.Immunol. 156:700; Stevens, et al. (1995). J. Immunol. 154:762;Rivoltini, et al. (1995). J. Immunol. 154:2257; Kawakami, Y. and S. A.Rosenberg. (1997). Int Rev Immunol. 14:173, by culturing these targetcells at 37° C. for 2 hours in 1 ml of medium containing 5 mg of peptideprior to labeling with ⁵¹Cr for use in cytolytic assays to demonstraterenewed susceptibility to specific T cell recognition.

In further instances, bulk and cloned TIL progeny were also assayedagainst autologous tumor (MU), allogeneic melanomas, as well as NK(K562), and LAK (Daudi), and EBV-transformed B lymphocyte targets: EBV-3(HLA-A1, B8, DR3), EBV-19 (HLA-A2, B18, DR5), using the foregoing⁵¹Cr-release assay. Pulsing included the following melanocytelineage-derived peptides: Tyrosinase (Rivoltini, et al., supra):MLLAVLYCL (SEQ ID NO:2) or YMNGTMSQV (SEQ ID NO:3), MAGE-3 (Gaugler, etal. (1994). J Exp Med 179:921): EBDPIGHLY (SEQ ID NO:4). Clones werescreened for cytotoxic activity at effector to target ratios of 50:1 andbelow.

PCR Analysis: Equal quantities of oligo-dT18 reverse-transcribed RNAswere subjected to RT-PCR analyses, as previously described (Kurnick, etal. (2001) J Immunol 167:1204), using multiple dilutions to establishconditions where initial amounts of control mRNAs resulted insub-saturating amounts of products, with representative templateconcentrations shown. Primers were designed from appropriate GenBankmRNA and genomic entries and designed to be intron-spanning to preventsimultaneous amplification of traces of genomic DNAs. Where this was notpossible RNAs were treated with RNase-free DNase I and repurified.

Primer sequences: (Forward {sense}/reverse{anti-sense} pairs) (SEQ IDNOs:5-22)

Melan-A/MART-1: CAAGATGCCAAGAGAAGATGCTCACT/ GCTTGCATTTTTCCTACACCATTCCA;β-Actin: GAGATCACTGCCCTGGCACCCA/ GCTCCAACCGACTGCTGTCACCTTCAC;gp100/Pmel17: CTGATTGGTGCAAATGCCTCCTTCT/ AGGAAGTGCTTGTTCCCTCCATCCA;tyrosinase: CAGCCCAGCATCATTCTTCTCCTCT/ GCAGTGAGGACGGCCCCTACCA;TRP-1: TGTTGCCCAGACCTGTCCCCT/ GCAACATTTCCTGCATGTCTTTCTCCA;TRP-2: CCTAGTGAACAAGGAGTGCTGCCC/ CGCTGGAGATCTCTTTCCAGACACAAC;MITF-M: TCTCTCACTGGATTGGTGCCACCT/ CATGCCTGGGCACTCGCTCTCTMITF-A: CCAAGCCTCCGATAAGCTCCTCCA/ CATGCCTGGGCACTCGCTCTCTGAPDH: TGAAGGTCGGAGTCAACGGATTTGGT/ CTGCAAATGAGCCCCAGCCTTCTMITF-M and MITF-A share a common reverse primerowing to their shared mRNA 3′ regions. PCR productidentities were confirmed by automated sequencing.

Example II

This example describes expression data of melanocyte-associated antigensand transcription factors.

Melan-A/MART-1 deficient cells, such as MU-X and EW, produce solublefactors that down-modulate antigen expression in otherwiseconstitutively positive cells (Kurnick, et al., supra);Ramirez-Montagut, et al., supra). To determine the natural repertoire ofgene expression of related proteins in a series of antigen-positive anddeficient cell lines, four Melan-A/MART-1-expressing melanoma celllines, 136.2, 453A, MM96L (an antigen-expressing variant, designatedMM96L+, and an Melan-A/MART-1 deficient variant designated MM96L−) andMU (an antigen-expressing variant, designated MU, and an Melan-AJMART-1deficient variant designated MU-X), and an additional five cell linesthat have weak or deficient Melan-A/MART-1 expression, MU-X, EW,IGR-39D, MM976L− and A375, as well as the Burkitt lymphoma-derived RAMOScell line, were studied (Table 1). Antigen expression of Melan-A/MART-1(MA/M1), gp100 and tyrosinase was assessed by cytoplasmic staining withappropriate monoclonal antibodies. In addition, assessment of grossdifferences in the relative mRNA steady-state levels for these markersbetween different cell lines was made following PCR amplification.

As shown in Table 1A, below, low expression of Melan-A/MART-1 isgenerally associated with low gp100 and tyrosinase expression. Among themelanomas, only EW secretes measurable amounts of protein (as determinedin ELISA), but an additional 5 cell lines show detectable OSM mRNAlevels, albeit weaker than EW (and non-melanoma RAMOS). Only MM96 andA375 appear to be deficient for OSM mRNA. Tyrosinase related proteinsTRP-1 and TRP-2 parallel the expression of the other melanocyte markers.

A series of transcription factors related to melanocyte differentiationwere also examined. As shown in Table 1B, the melanocyte-associatedallele of MITF, namely MITF-M, was expressed strongly on theMelan-A/MART-1 expressing tumors, but not on the antigen deficient celllines, except for A375. In contrast, the MITF-A isoform was expressed onall but the RAMOS cell line. Sox 10 showed a pattern similar to MITF-M,although it was detectable in MU-X as well as A375. Pax 3, brn2 and tbx2were widely expressed among all of the melanomas, although tbx2 was onlyweakly expressed in EW.

TABLE 1 Antigen and Transcription Factor mRNA Levels in Melanoma CellLines. TUMOR 136.2 453A MM96L MU-89 MU-X EW IGR-39D A375 RAMOS 1A.Melanocyte Lineage Antigen Expression (Protein and mRNA) OSM ++ + − +++/− ++++ ++ − ++++ MA/M1 ++++ ++++ ++++ ++++ +/− +/− +/− +/−* − gp100++++ ++++ ++++ ++++ + + + + − tyrosinase ++++ ++++ ++++ ++++ − − +/− +/−− TRP-1 ++++ ++++ ++++ +++ ++ − ++ ++ − TRP-2 +++ ++++ ++++ ++ +/− − −++ − *A375 have detectable mRNA for Melan-A/MART-1, but are relativelydeficient in cytoplasmic protein expression. 1B. Transcription FactorExpression (mRNA) MITF-M ++++ ++++ ++++ ++++ +/− +/− +/− ++ − MITF-A++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ − BRN2 ++++ ++++ ++++ ++++ ++++++++ ++++ ++++ − STAT3 ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ Pax3++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ + SOX 10 ++++ ++++ ++++ ++++ + −+/− ++++ − Tbx2 ++++ ++++ ++++ ++++ +++ + +++ ++++ − The (++++)designation indicates easily detectable (relatively high level) productformation. Where the designation of +/− is assigned, product levels werereproducibly low, often requiring a second round of nested PCR forunequivocal detection. (Comparison of the relative levels betweenseparate markers is not feasible with these assays).

Example III

This example describes down-regulation of melanocyte-associated antigensMelan-A/MART-1 and gp100. This example also describes data indicatingthat IFN-beta up-regulates melanocyte-associated antigens Melan-A/MART-1and gp100.

Expression of Melan-A/MART-1 can be down-regulated by culture withsupernatants from Melan-A/MART-1-negative tumors such as EW and A375(Ramirez-Montagut, et al., supra). In brief, MU tumor cells werecultured for 3 days in control medium or in 20 ng/ml of OSM (FIGS. 1Aand 1D), or in supernatants from EW (contains OSM) (FIG. 1B) or A375tumor cells (does not contain OSM) (FIG. 1C). Cells were stained forcytoplasmic expression of Melan-A/MART-1 protein (FIGS. 1A-1C) or gp100(FIG. 1D) and assayed by flow cytometry.

The data indicate that Melanoma Antigen Silencing Activity (MASA)produced by EW cells includes OSM and at least one additional solublefactor, designated MASA2, that is present in EW supernatants followingremoval of OSM, and is also present in A375 cells that do not produceOSM.

The loss of Melan-A/MART-1 is associated with a marked diminution in theability of T cells to lyse tumor cells which have been treated withMASA-containing supernatants (Ramirez-Montagut, et al., supra). The lossof T cell-mediated lysis can be overcome by the addition of theMelan-A/MART-1-derived peptide, AAGIGILTV (SEQ ID NO:1), which restorescytolytic susceptibility. Loss of Melan-A/MART-1 is generallyaccompanied by diminished gp100 and tyrosinase, as well as othermelanocyte lineage proteins, indicating that there is a “global” changein the tumor cells. However, the down-modulation of antigen expressionappears to be somewhat selective as the HLA Class I antigen needed forpresentation of the melanoma peptide is not down-modulated (Kurnick, etal., supra). When MASA-containing conditioned medium was removed fromthe Melan-A/MART-1 expressing tumor cells, there was renewed expressionof this antigen. These antigen positive cells are again lysed byMelan-A/MART-1-specific cytotoxic T cells.

Oncostatin M and other melanoma cell line derived factors can downmodulate melanocyte lineage antigen expression in various melanoma celllines (Kurnick, et al., supra). A number of cytokines for up anddown-modulating activity of melanocyte lineage antigens were evaluated.

Surprisingly, interferon-beta had up-modulating activity on all melanomacell lines, both low and high expressors of Melan-A/MART-1 (FIG. 2).Furthermore, interferon-beta could reverse the down modulating effect ofOncostatin M on gp 100 (FIG. 3—HMB 45 staining), and the effect ofinterferon-beta was augmented by treating the cells with a DNA methylaseinhibitor such as 5 azadeoxycytidine (FIG. 4—gp 100 (HMB) staining).

In sum, the foregoing data indicate that interferon-beta can inhibit theantigen down-modulating effect of Oncostatin-M, a known cytokine capableof mediating antigen-silencing in the melanoma system, as well asdown-modulation induced by an additional molecule or molecules producedby melanoma cells (MASA) that manifest antigen silencing.Interferon-beta can up-regulate Melan-A/MART-1 antigen expression on allmelanoma cell lines studied to date regardless of the mechanismcontrolling antigen expression down-modulation.

IFN-β also enhances expression of MHC class I antigens (HLA-A,B and C),and IFN-γ enhances both class I and class II MHC antigens, thusincreasing production of antigen-presenting molecules on tumor cells.Expression of new TAA and new HLA is therefore a doubly-effectivetreatment for enhancing T cell recognition of tumor cells, making itmore likely that a cytotoxic T lymphocyte (CTL) will bind and kill tumorcells treated with IFN-β.

Example IV

This example describes down-regulation of melanocyte-associated antigensMITF, tyrosinase, TRP-1 and TRP-2. This example also describes dataindicating that transfection of MITF-M up-regulated Melan-A/MART-1antigen expression.

Tumors with low or absent Melan-A/MART-1 are also relatively deficientin tyrosinase and gp100; 3 of 4 low-Melan-A/MART-1 melanomas have lowMITF-M, including the MU-X line derived from Melan-A/MART-1+MU cells.The sox10 regulator of MITF-M expression is deficient in 2 of 4 of thelow-Melan-A/MART-1 melanomas, while another melanocyte-lineagetranscription factor, tbx2, was deficient at the mRNA level only in theMelan-A/MART-1-low EW cell line (Table 1).

OSM induces down-modulation of various melanocyte-related genes,including Melan-A/MART-1, tyrosinase, gp100, TRP-1 and TRP-2 (FIG. 5).While OSM also down-modulates MITF-M expression, the MITF-A isoform isnot detectably responsive to OSM. Expression of the microphthalmia genevariants is dependent on different promoters and with differentN-termini in their respective translated proteins (Udono, et al. (2000).Biochim Biophys Acta 1491:205). The differential action can provideclues to the promoter elements responsive to OSM; for example, only theMITF-M isoform promoter has a perfect CRE site.

All four of the Melan-A/MART-1 deficient melanoma cell lines studiedproduce strong antigen-silencing activity. This suggests a correlationbetween antigen expression and the production of an antigen-silencingfactor. Melanocytes, which normally express this antigen, must bedown-regulated in order to shut off transcription of this protein. If atumor mutant had lost the Melan-A/MART-1 gene, or its promoter, therewould be no selective advantage for the cell to continue to produce an“antigen-silencing” factor. The simultaneous loss of tyrosinase andgp100 suggest that any mutations in these cells would be targeting somegene regulatory molecules, as it would be less likely that all of thesechromosomally distinct genes would be deleted or mutated simultaneouslyin several different tumor lines. Whether such a gene is involved indifferentiation of the melanocyte lineage, or perhaps maintenance of aless mature phenotype, active production of MASA seems to becharacteristic of antigen-negative melanomas.

To express MITF-M in cell lines expressing low levels of Melan-A/MART-1,MITF-M coding sequence was amplified from MITF-M-positive cells andcloned in an SV40-promoter expression vector (pSV21ink); translation ofthe MITF-M insert uses optimal Kozak initiation signals (Kozak (1999).Gene 234:187). Constructs were transfected into low-Melan-A/MART-1expressor melanoma (MU-X and A375). In all studies controls comprisedempty vector, transfection reagents in the absence of added DNA, andcorresponding untransfected cells. Data shown in FIG. 6 for A375 andMU-X tumor cells transfected with MITF-M for 24 hours in the presence(10 μM) or absence of U0126 before PCR amplification of Melan-A/MART-1.

MUX and A375 cell lines exhibited up-regulation of endogenousMelan-A/MART-1 after transfection with the MITF-M expression construct(FIG. 6). A MEK inhibitor (U0126) was then added to determine whether itcould synergize with ectopically introduced MITF-M. In this regard,plasmid-encoded MITF-M gene is not subject to the normal MITF-Mtranscriptional controls, since U0126 down-modulates native MITF-Mmessage.

U0126 addition augmented enhancement of Melan-A/MART-1 expression inboth MITF-M transfected A375 and MU-X tumor cell lines. These resultsindicate that controlling MITF-M expression would also controlMelan-A/MART-1 expression.

Example V

This example describes data indicating that IFN-β up-regulation ofmelanocyte-associated antigen expression increases T cell killing ofmelanoma cells.

In brief, A375 cells were treated with 100,000 units of IFN-β for threedays. The cells were subsequently labeled with ⁵¹Cr and tested astargets in a cytotoxicity assay using bulk anti-melanoma T lymphocytesas the effector cells (Example I).

As shown in FIG. 7, up-regulation of antigen expression induced by IFN-βresults in melanoma cells that can be killed by T lymphocytes. Theseresults demonstrate that IFN-β can increase targeting of tumor cells bythe immune system.

Example VI

This example describes recombinant constructs used for screeningcompounds which effect tumor-antigen expression.

To identify other compounds having the same effect as interferon-beta,recombinant DNA constructs which contain a sequence tag (e.g luciferase,or green fluorescent protein (GFP) or an enzyme activity) linked to aMelan-A/MART-1 regulatory element (e.g., promoter) can be constructedand inserted into Melan-A/MART-1 melanoma cells (e.g., a low expressorcell line). Transfected cell lines can then used for screening of smallorganic compounds and larger compounds having biological activity, e.g.,compounds that up-regulate expression of Melan-A/MART-1, and otherantigens.

For identification of TAA modulating agents, a reporter thatincorporates the promoter region from the Melan-A/MART-1 melanocytelineage differentiation antigen and tag sequence was constructed. Theexemplary construct including green fluorescent protein (GFP) isillustrated in FIG. 8. GFP reporter systems have been previouslydescribed (Haseloff, (1999). Methods Cell Biol 58:139; Tsien (1998).Annu Rev Biochem 67:509; Chiesa et al. (2001). Biochem J 355:1; Belmont(2001). Trends Cell Biol 11:250).

A number of melanoma cells have been transfected with linearizedconstructs expressing GFP from an extended Melan-A/MART-1 promoter (1176bp) and separately with a construct expressing GFP by means of the SV40promoter (depicted in FIG. 8).

Stable transfectants were selected by co-transfection with a plasmidconferring resistance to Geneticin (G418). The expression results ofsuch constructs are shown in FIG. 9.

Melan-A/MART-1 promoter (SEQ ID NO: 26)AGATCCTGCCACTGCACTCCAGCCTGGGCGACAGAGTGAGTCTCCATCTCAGAAAAAAAAAATGTGTTTGAGCCTAGTTATAATGATTTAAAATTCATGGTCCGACACCGCAATTACTTTTGCACCAACCTAATTGATGTCTAAGTAGGTCATATTCTACCTGCAAAAAGAAAATTTCATCTATCCCTTTCACATAGATGGAAACCCACTATCTCCAGTGGACAGTTAACACCAAAGGCATCACAGAGAACTCATGGAGCTCAGCTGAGGAGGTTTCAGGGATTTTTCTATTTCCTTTTCTTGATTATGAGAGTCTGGGACTAGATGCTCTCCAGACCTGTGCCTAAAGACTCTTCAACCCTTTGAGATGGAGATGAGGGAGGGAATAGGGAACCCAGTTTAGTTTGGATTTCAGATCCTTTTGTGGGTCATAAGCGTGATGATTGGGTTTCCATGTTCACGTGTGAGATATGCCTCCCTCAAACCTTGTTACAATGACATGGGCACCTTACCTATCTGACATGAGAAAAACAAATGTGGATTTCAGATAAACAAAAAATAACTCTTTTAGTGTATATGTCCCATAGAATATGTGGACATATTTATCCTAAAAATATTGTATGGGACATAGTTGTATTAAGAAACTGTTCATTGTTTATCTGAAGTTCAAATTTAACTGGGCATCCTCCTCAGCTGAGCTCCATGAGTTCTCTGTGATGCCTTTGGTGTTAACTGTCCACTGGAGATAGTGGGTTTCCATCTATGTGAAAGGGATAGATGAAATTTTCTTTTTGCAGGTAGAATATGACCTACTTAGACATCAATTAGGTTGGTGCAAAAGTAATTGTGGTGTCGGACCATGAATTTTAAATCATTATAACTAGGCTCATGTCATATTTTATGTGACATGGCAATCCTATGGAGGAGGGACCAACATTTAAAATAAATGGCTTCCCTAGGATAGAGCACTGGGACTGGGGAAAACAGAGGCCACAGTCAGCTGTGACTTTTTGAAGGAAGGAATAAAGTTGGTTTCTTTCATGCCAATTTAGCAATTACAGACGACCCCGTCAGAAATCTAAACCCGTGACTATCATGGGACTCAAAACCAGGAAAAAAAATAAGTCAAAACGATTAAGAGCCAGAGAAGCAGTCTTCATACACGCGGCCAGCCA GFP coding sequence (SEQ ID NO: 27)ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCA TGGACGAGCTGTACAAGTAASV40 late polyA signal (SEQ ID NO: 28)CAGACATGATAAGATACATTGATGAGTTTGGACAAACCACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGGTGTGGGAGGTTTTTTAAAGCAAGTAAAACCTCTACAAATGTGGTA

The above sequences are the relevant functional portions of reportertransfected into the appropriate cell lines such that GFP can beexpressed from the Melan-A/MART-1 promoter (in cells with the righttranscriptional apparatus; i.e. “high” Melan-A/MART-1 cells). An“extended” MART promoter (1176 bp) was derived from amplification ofhuman genomic DNA with primers corresponding to the 5′ and 3′ ends ofthe sequence as shown. GFP sequence (“EGFP”) is from a Clontech vector.Initiation codon is underlined; termination TAA codon at end of thissegment. SV40 late poly A signal: SV40 sequences are widely used and areknown in the art. No intronic sequences are present in the construct.

In brief, both high and low antigen expressing cells, MM96L and A375,respectively, were transfected with the exemplary Melan-A/MART-1-GFPrecombinant reporter construct (FIG. 8). After cloning out cellscontaining the construct, the effect of interferon-p was studied.

The MM96L-Melan-A/MART-1-GFP reporter cells treated with IFN-β for 72hours showed augmentation of GFP fluorescence (GFP emission is shown inFIG. 9), in common with its endogenous Melan-A/MART-1 gene. A375reporter cells treated with IFN-β also showed augmentation of GFPfluorescence. In contrast, SV40 promoter-driven GFP exhibited no suchresponse. This data therefore demonstrated that the GFP reporter systemsrecapitulated the regulation of native Melan-A/MART-1 gene. A reporterdriven by Melan-A/MART-1 regulatory region cellular system is thereforeuseful for screening and identifying compounds, agents and drugs thatup-regulate antigen expression.

These reporter constructs can be employed in vivo. For example, tumorcells can be propagated subcutaneously in immunodeficient mice, andinduced with IFN-β in vivo. The tumors can be injected directly withantigen-up-regulators (e.g., IFN-β, assuring that there is local drugavailable. Mice can also be treated with IFN-β subcutaneously followingestablishment of antigen negative tumor cells (such as MU-X and A375).By biopsying subcutaneous tumor sites at regular intervals followingIFN-β therapy, a time-course for induction of antigen will be developed.Any reversion of tumor cells to antigen-negative status followingtermination of drug therapy can also be studied. Also, since human Tcells that are able to recognize and lyse antigen positive tumors areavailable, tumor biopsies stained with antibody to human CD3 candemonstrate altered infiltrating of adoptively transferred human CTLfollowing antigen-induction therapy. In particular, observing therecruitment of T cells to tumors that are expressing GFP, as opposed tothose that are GFP negative, as a demonstration of induced tumor antigen(Melan-A/MART-1). The studies using GFP-transfected tumor cells willparallel those for the un-transfected cells described below.

Example VII

This example describes in vivo applications of IFN-β and tumorassociated antigens (TAAs). This example also describes exemplary assaysfor monitoring the effect of IFN-β alone and in combination with TAAs.IFN-β is safe and well-tolerated in ambulatory patients, thus providingan agent with relatively well-described in vivo toxicities andtolerances. By combining IFN-β therapy with tumor-associated antigens,both enhanced immunity and enhanced tumor antigen expression leading tomore effective tumor killing in vivo are expected. T cell immunity andtumor antigen expression during in vivo administration, and correlatingclinical responses with the induction of T cells specific for tumorantigens, as well as with antigen expression by the tumors, before,during and after therapy has been instituted will be analyzed.

Human tumor xenografts in mice will enable evaluation of the in vivoinduction of tumor (e.g., melanocyte) antigens. Both antigen-positiveand antigen-negative tumor cells can co-exist in human tumors that havedeveloped spontaneously over a period of months to years. Tumorheterogeneity is not readily demonstrable in tumor transplant modelswhere the tumor-injected animals are inherently short-lived, andgenerally the tumors are clonally homogeneous during the course of thestudies. Thus, although tumors in animal models may not absolutelyreflect tumors in humans, there are compelling reasons to develop animalmodels employing low antigen expressing xenografts. For example,prognosis for a melanoma patient with established metastatic disease isquite poor even with aggressive current therapies, and the use ofimmunotherapies alone have shown limited success even when ratherinnovative treatment regimes have been employed.

Animal treatments include combining multi-epitope tumor (e.g.,melanocyte) antigen with IFN-β therapy to induce enhanced host immunityagainst the tumors, or to maintain and increase tumor antigen expressionto enhance recognition of tumor cells which might otherwise escapeimmune destruction. Clinical endpoints include assessment of both hostimmunity and expression of tumor antigens, achieving improved hostimmunity (systemic and intra-tumoral cellular and huimoral immunity;Melan-A, MAGE-10, and NY-ESO-1b specific CD8⁺ T cells—measured bytetramer method, Melan-A, MAGE-10, and NY-ESO-1b specific activated(interferon gamma releasing) CD8⁺ T cells—measured by ELISPOT; DTH totyrosinase leader, Melan-A, MAGE-10, and NY-ESO-1b peptide; NY-ESO-1reactive antibodies, etc.) or inhibition of tumor growth or tumordestruction (measurement of tumor size) in patients with tumors, such asmelanoma; and toxicities and adverse events (as defined by NationalCancer Institute Common Toxicity Criteria (CTC) Scale).

TAAs used are components of proteins recognized by the autologous immunesystem on tumors such as melanomas. One of more TAAs could be expressedin the tumor. Expression of tyrosinase, Melan-A, NY-ESO-1, LAGE, andMAGE-10 in tumor tissue can be tested by reversetranscription-polymerase chain reaction (RT-PCR) analysis orimmunohistochemistry. As all study peptides are presented by HLA-A2,patients expressing HLA-A2 are treatment candidates.

Enhanced immunity to the antigen Melan-A has been observed whenMontanide ISA™51 is used as an adjuvant with Melan-A peptide. Theaddition of Montanide ISA™51 to TAAs given with or without IFN-β islikely to lead to enhanced immunological and clinically beneficialeffects in melanoma patients.

Exemplary TAAs, formulations and routes of administration are asfollows:

Tyrosinase leader: HLA-A2 binding peptide encoded by tyrosinase gene;sequence

-   -   MLLAVLYCL (SEQ ID NO:2); position 1-9    -   Formulation: 333 μg/mL tyrosinase leader in 100% DMSO    -   Intended dose: 100 μg    -   Vial size: 1-mL vial with 0.45 mL peptide solution    -   Route of administration: intradermal    -   Source: LICR        Melan-A ELA: Analog of HLA-A2 binding peptide encoded by Melan-A        gene; sequence    -   ELAGIGILTV (SEQ ID NO:23); position 26-35 (E27L)    -   Formulation: 333 μg/mL peptide in 30% DMSO in phosphate buffered        saline    -   Intended dose: 100 μg    -   Vial size: 1-mL vial with 0.45 mL peptide solution    -   Route of administration: intradermal    -   Source: LICR        MAGE-10.A: HLA-A2 binding peptide encoded by MAGE gene; sequence    -   GLYDGMEHL (SEQ ID NO:24); position 254-262    -   Formulation: lyophilized powder    -   Intended dose: 300 μg    -   Vial size: 1-mL vial with 400 μg peptide    -   Route of administration: intradermal    -   Source: LICR        NY-ESO-1b: HLA-A2 binding peptide encoded by NY-ESO-1 gene;        sequence    -   SLLMWITQC (SEQ ID NO:25); position 157-165    -   Formulation: 2 mg/mL NY-ESO-1b in 100% DMSO    -   Intended dose: 100 μg    -   Vial size: 1-mL vial with 0.3 mL peptide solution    -   Route of administration: intradermal    -   Source: LICR

Montanide ISA-51

-   -   Formulation: montanide oleate (Montanide 80) in mineral oil        solution (Drakeol 6VR)    -   Intended dose: 1.0 mL    -   Vial size: 3 mL    -   Route of administration: subcutaneous    -   Source: SEPPIC, Paris, France

Interferon β

-   -   Rebif® 22 ug (6×10⁶ IU)/vial Serono, Rockland, Mass.

Exemplary patient inclusion criteria include one or more of thefollowing, for example, confirmation of metastatic melanoma; HLA-A2positive; Relapsed Stage 1V melanoma with lesions that are resectable oraccessible to biopsy; at least 4 weeks since surgery before initiatingprotocol; at least 4 weeks since the last chemotherapy, biologictherapy, or immunotherapy; no concurrent biologic therapy orimmunotherapy; performance status >70 (Karnofsky Scale); and lifeexpectancy ≧4 months.

Exemplary laboratory values of candidate patients can be within thefollowing limits:

Hemoglobin ≧9.0 g/dL ≧10.0 g/dL (if <50 kg) Neutrophil count ≧1.5 ×10⁹/L Lymphocyte count ≧0.5 × 10⁹/L Platelet count ≧100 × 10⁹/L Serumcreatinine ≦1.8 mg/dL Serum bilirubin ≦2 mg/dL

Optional exemplary patient exclusion criteria include one or more of thefollowing, for example, clinically significant heart disease (NYHA ClassIII or IV); serious illnesses, eg, serious infections requiringantibiotics, bleeding disorders; prior bone marrow or stem celltransplant; history of immunodeficiency disease or autoimmune disease;metastatic disease to the central nervous system, unless treated andstable; HIV positive; chemotherapy, radiation therapy, or immunotherapywithin 4 weeks before study entry (6 weeks for nitrosoureas);concomitant treatment with steroids, antihistaminic drugs, ornonsteroidal anti-inflammatory drugs (unless used in low doses forprevention of an acute cardiovascular event or for pain control)—topicalor inhalational steroids are permitted; participation in anotherclinical trial within 4 weeks prior to enrollment; pregnancy orlactation; women of childbearing potential not using a medicallyacceptable means of contraception; unavailability of the patient forimmunological and clinical follow-up assessment.

For melanoma, an exemplary protocol employs one or more of four TAApeptides (melanoma peptide vaccine) comprising a tyrosinase leader,Melan-A ELA, MAGE-10.A2 and NY-ESO-1b. Peptide(s) will be administeredby subcutaneous injection every 3 weeks for six vaccinations. Peptideswill be mixed together with Montanide ISA-51 and given at separateinjection sites. In addition, patients will be randomized to receive ornot to receive IFN-β by subcutaneous injection, 3 times weekly (M, W F)(6 million units per injection of IFN-β) for each of the three weeksbetween the vaccine boosts, beginning at week 7 (i.e. with the thirdvaccine injection). This protocol will allow for primary and earlysecondary immune responses to be initiated prior to introducing an agentthat is unlikely to alter effector phase immune responses, but mightalter the cytokine repertoire during initial vaccine induction ofanti-tumor immunity. Waiting for an early immune response to developminimizes the time for IFN-resistant tumors to be selected before theimmune response has been sufficiently enhanced to destroy tumors havingup-regulated antigen expression.

Patients can be monitored for toxicity after each vaccine and IFN-βinjection. Systemic immunity can be assayed using blood samples to beobtained at baseline and at specified time points, for the assessment ofpeptide-specific antibodies by ELISA, as well as peptide-specific CD8⁺ Tcells by tetramer analysis and ELISPOT. Peptide-specific delayed-typehypersensitivity (DTH) skin reaction will be measured at baseline andafter the third and sixth set of peptide injections. If DTH reactionsoccur at other time points, they will be measured. Tissue samples fromone metastatic lesion will be obtained at baseline and at least one timeafter two cycles of interferon β treatment for evaluation of antigenexpression. Additional tests for peptide-specific cellular and humoralimmunity will be done two weeks after the sixth set of peptideinjections. Clinical hematology and biochemistry measurements will betaken at baseline, and as specified in the protocol schema. Diseasestatus will be assessed at baseline and two weeks after the sixth set ofpeptide injections.

Whenever accessible tumor deposits are available, and can be biopsied,or excised with minimal risk to the patients being treated, bothintra-tumor immunity and histology and antigen expression on tumor cellswill be investigated. 3 types of tests will be performed wheneversufficient tissue is available to allow for the following assays:

Histology and antigen expression on tumor cells: Routine histology willbe performed to evaluate tumor necrosis and the status of infiltratinglymphocytes. Frozen sections of tumor tissue will also be stained forexpression of the antigens to determine both intensity and heterogeneityin antigen expression, particularly with respect to any regressing orprogressing lesions. In addition to evaluation of tumor and host immuneresponses, image analysis of tumor antigen expression andmicro-dissection specimens for amplification of mRNA for quantitativePCR analysis on tumors before and after therapy will be conducted.

Image Analysis: In order to evaluate enhanced MHC and melanocyte antigenexpression, biopsies will be stained with antibodies to HLA Class I andII antigens, as there should be an increase in MHC expression if thetumor cells are responsive to IFN-β. In parallel, the tissues will bestained with antibodies to the tumor-associated antigens (Melan-A,Tyrosinase, N.Y.-ESO and MAGE-10). Both immunoperoxidase staining andFITC-fluorescent-tagged antibody staining will be performed to acquirequantitative data on the levels of antigen expression in the tissue as awhole, and tumor cells individually.

Molecular Analysis of single tumor cells present in biopsies posttherapy: In addition to conventional histological techniques, usinglaser capture micro-dissection technology, individual tumor cells willbe evaluated for expression of a larger series of melanocyte associatedantigens and transcription factors to determine not only which of thevaccine antigens are expressed, but also to determine if there is moreconsistent expression of additional melanocyte lineage antigens that ismore amenable to targeting in subsequent treatments. Inclusion of thefollowing genes (Table 2) will allow evaluation of improvedimmunotherapy protocols should additional antigens prove to be moreamenable to homogeneous expression either with or without additionalinduction by IFN-β. In addition to the choice of genes encoding thevaccine antigens and HLA-A2, selection of the panel genes is made on thebasis of their relevance to the melanocytic lineage, known role incontrolling melanocytic gene expression, relevance to the IFN-βresponse, and as control markers.

At the single cell level correlations between levels of mRNAs expressedfrom antigen genes and those expressed from chosen transcription factorgenes during the course of the treatment can be evaluated. MITF-M isstrongly associated with the control of expression of a number ofmelanocytic antigens including tyrosinase and Melan-A/MART-1. SOX10 isone transcription factor in turn regulating MITF-M, and which is notexpressed in some of the low antigen-expressing cell lines. Type Iinterferons (including IFN-α and IFN-β) use a common receptor composedof two subunits. Examining expression of other antigen genes in additionto those included in the vaccine preparation will be performed asexpression of melanocytic antigens is regulated coordinately. Up- ordown-regulation of Melan-A/MART-1, for example, is often correlated witha corresponding change in TRP-1, TRP-2, and gp100 expression.

Evaluation of biopsy material from treated patients to determine whichantigens are still expressed, and which are enhanced by IFN-β will tohelp evaluate tumor heterogeneity, and more importantly, homogeneity ofantigen expression that can be utilized for identification of targetsthat will make immunotherapy a more successful approach.

TABLE 2 Exemplary genes to be evaluated for expression in tumor cells.Gene Classification Gene Name Antigens in vaccine Melan-A, tyrosinase,MAGE1-A2, NY-ESO-1b HLA HLA-A2 Other Melanoma-Associated Antigens TRP1,TRP2, gp100 (pmel 17) Melanoma Associated Transcription MITF-M, SOX10Factors IFN-Type I Receptor IFNAR-1, IFNAR-2 Other Markers MITF-A,β-Actin

TaqMan chemistry and appropriate instrumentation allows rigorousquantitative PCR analysis of mRNA levels of desired molecular targets,and has been applied towards single-cell analyses. To obtain informationregarding expression of a panel of markers, some of which may be at lowcopy number per individual cell, an amplification step from eachsingle-cell mRNA source will be performed, where it is critical thatsuch a step faithfully preserves the relative abundance of each specieswithin the mRNA transcriptome. With single or low numbers of cells, T7RNA polymerase-mediated amplification via the generation ofcomplementary RNA transcripts (cRNAs) (Eberwine. (1996). Biotechniques,20: 584; Luo, et al., (1999). Nat Med, 5:117; and Abe, et al., (2003). JHum Genet, 48:142) can generate long in vitro transcripts (Riechmann, etal., (1990). Virology, 177:710; Puurand, et al., Virus Res, 40:135,1996; and Shi, et al., (2002). J Virol, 76:5847), well in excess of theMITF mRNAs. Following T7 polymerase-mediated amplification, theresulting cRNAs are reverse-transcribed with random hexamers forsubsequent TaqMan Q-PCR analysis.

The “housekeeping” genes commonly used for normalization purposes in avariety of expression-based studies β-actin, GAPDH) have been noted asproblematic for tissue-based and single-cell studies. Thus, apresynthesized internal spiked standard in the assays, in the form of asurrogate non-mammalian mRNA (luciferase) generated by in vitrotranscription, will be added. This is achieved by cloning luciferasecoding sequence into Promega Corp. vector pSP64polyA, and preparingpolyA+ run-off in vitro transcripts with SP6 polymerase. The plasmidtemplate is digested with RNase-free DNAse, the RNA transcripts purifiedby three cycles of ammonium acetate precipitation, quantitatedspectrophotometrically and gel tested for full-length integrity. Ifnecessary, full-length species will be purified by excision of thecorrect gel band and extraction from agarose. A quantity equivalent to100 copies of polyA+ luciferase RNA will be added to each cell lysateprior to initial reverse-transcription, second-strand cDNA synthesis andsubsequent T7 polymerase amplification of cRNA. In consequence,detection of the internal introduced standard (with its own specificprimer/probe TaqMan system) will have identical enzymatic requirementsas for the cellular mRNAs themselves. Levels of each target gene in theabove panel will then be expressed as ratios to the levels of theintroduced standard. β-actin (high abundance mRNA) and MITF-A (moderateto low abundance mRNA) is included in the gene panel for single-cellanalysis (Table 2) as widely-expressed controls for confirming that theendogenous mRNAs themselves from each cellular isolate are intact.Normalization will more accurately use the introduced surrogate mRNAstandard.

In addition to analyzing immunity represented in the circulatinglymphocytes in the blood, intra-tumoral lymphocytes with tetramers willbe stained to determine the frequency of peptide-specific CD8+ T cellspresent within the tumor tissue. Furthermore, by culturing small tumorfragments in the presence of Interleukin-2, large numbers of invivo-activated tumor-infiltrating lymphocytes can be further studied forcytotoxic activity against tumor targets (Hishii, et al., (1997). Proc.Natl. Acad Sci (USA), 94:1378; Ramirez-Montagut, et al., (2000). 119:11;Kradin, et al., (1989). Lancet, 1:577; Hishii, et al., (1999). Clin ExpImmunol, 116:388; and Pandolfi, et al., (1991). Cancer Res., 51:3164)from the same patient when available, and from HLA-A2 matched cell linesif autologous tumor target is unavailable. Functional assessment ofcytotoxic activity will complement the tetramer assays, which will givean indication of the frequency of T-cell receptor positive cells withspecificity for the tumor vaccine antigens. These studies will indicatewhether TAAs administered with IFN-β, increase local tumor immunity forsuccessful tumor immunotherapy.

Although it is anticipated that there will be a measure of tumor antigenheterogeneity in tumor biopsies, both antigen positive andantigen-deficient tumor cells can show enhanced tumor antigen expressionfollowing treatment with IFN-β. Evaluating the ability of tumor toup-regulate both melanocyte lineage antigens and HLA antigens willreveal whether individual tumor deposits contain IFN-responsive tumorcells. In the event tumor cells show no antigen induction, thepossibility of lost IFN-receptors, or lost IFN-response elements wouldbe expected to limit the efficacy of antigen-up-regulation therapy.

The combined therapy (e.g, IFN-beta and TAAs) will enhance clinicalresponses in tumor (e.g., melanoma) patients via enhanced antigenexpression, improved cell-mediated immunity and destruction of tumorcells with antigen expression. To the extent that tumor remains aftertherapy, evaluation of tumor antigen expression and host immune responsein situ will allow refinements in the treatment protocol. For example,if there is loss of TAA expression that is present in the vaccine, butretention of other TAAs on the tumor cells, a follow-up administrationcould be performed using different TAAs to which T cells can betargeted. Also, if TAAs not represented in the original vaccine areup-regulated with IFN-β, future administrations can include such TAAsresponsive to up-regulation.

Tissue Processing and Analysis: For tissue sample processing, lasercapture microdissection (LCM) has emerged as a revolutionary techniquefor genetic analysis, combining precise microscopy with molecularexpression profiling at the single cell level (Emmert-Buck, et al.,(1996). Science, 274:921; Schutze and Lahr, (1998). Nat Biotechnol,16:737; Sgroi, et al., (1999). Cancer Res, 59:5656; Miura, et al.,(2002). Cancer Res, 62:3244; De Preter, et al., (2003). Cancer Lett,197:53; and Fend and Raffeld, (2000). J Clin Pathol, 53:666). The sameprocessing scheme towards single-cell analysis of archived samples ofprimary resected tumor samples from each of the patients in the studywill be applied. Preserved paraffin-embedded materials can be used assources of such material by means of laser-capture microdissection.

For each patient biopsy sample LCM 20 single cell isolates withmorphological characteristics of melanoma tumor cells will be obtained.Subsequently, with the procedure described above, quantitativefluorogenic PCR with the TaqMan chemistry as described (Xiang, et al.,(2001). Immunol Cell Biol, 79:472) will be performed using triplicatedeterminations in each case. To improve the screening rate and forreasons of economy, the 384-well plate format now available with theTaqMan instrumentation will be employed. Primers and probes will bedesigned with PrimerExpress software, with the primers positioned suchthat they span large introns if possible (this is feasible in allcases). In any case, owing to the cRNA amplification step, it isunlikely that the minimal amount of genomic DNA contributed by theoriginal target cell will be a confounding factor for expressionanalysis. Preliminary studies will define optimal probe concentrationsfor each primer/probe combination. Also, preliminary work will beperformed to determine the assay sensitivity achievable with the cRNAamplification under the conditions. In practical terms, this means theamount of total reverse-transcribed cRNA needed for accurate Q-PCR.Since >1000-fold amplification with the T7 RNA polymerase is readilyachievable in even a single round (Eberwine, (1996), supra), limitationsfrom the amounts of amplified target cRNA is unlikely.

Mouse Models Murine tumor models, developed in an immunodeficient mouse,will provide a system to develop or evaluate assays for monitoring thehuman clinical trial as well as testing the efficacy of IFN-β toup-regulate antigen expression in vivo. This work will afford anopportunity for comparison of the responses in both human clinical trialand the in vivo mouse model.

Human tumor cell lines will be propagated in culture and implanted intorag 2-deficient (rag-2^(−/−)) mice. When rag2^(−/−) mice are challengedwith 1×10⁶ melanoma cells, palpable tumors are apparent within 2 weeksand these tumors reach an approximate area of 200 mm² within 4 weeks. Inbrief, rag^(−/−) mice will be injected in the s.c space with 1×10⁶melanoma cells. When tumors reach a size of 100 mm², mice will berandomly assigned to groups of 5 for treatment. “Control” animals willbe treated with an injection of compound diluent. ‘Protocol’ animalswill be treated with compounds using escalating doses reflective ofprevious reports (Clemons, et al., (2002). Pancreas, 25:251) (serumlevels of IFN-β will be monitored by ELISA). Treatments will becontinued every other day for one week. Every other day for 7 days, micewill be sacraficed and tumors excised and evaluated. Each tumor will bedissociated using collagenase and dispase solutions. The resultingsingle cell suspension will be used for flow cytometric or PCR analysisof antigen expression as with in vitro cultured cells. Each set ofstudies will be repeated twice.

High and low antigen expressing tumor cells, MU and MU-X, cultured inindividual mice will be subjected to fine needle biopsies to providecells for single cell PCR and immunohistochemical experimentation.Expression of mRNA for the genes listed in Table 2 will be evaluated bythe same single-cell Q-PCR procedure as described above.

Immunodeficient mouse models will be used to evaluate the ability ofantigen-enhancing agents to up-regulate tumor antigen expression invivo. Multiple antigen induction observed in human melanoma cells invitro will be evaluated in vivo. Bio-availability of IFN-β in animaltumor models, using doses of antigen up-regulatory agents that will besub-lethal to the mouse, will be determined. Both MU and MU-X tumorcells can be grown in immunodeficient mice in subcutaneous sites(Fukumura, et al., (1995). Cancer Res, 55:4824). These studies willallow refinements to human clinical trial described above, as regardsimmunohistochemistry and single cell rtPCR evaluation of antigenexpression.

A typical dosing efficacy protocol is described below for comparing theresponse of antigen positive (MU) and antigen negative (MU-X) tumorcells. In each case tumors will be stained with antibodies toMelan-A/MART-1 (A103), gp100/pme117 (HMB45) and HLA Class I antigen(W6/32). In addition, RNA will be extracted for PCR assessment ofinduction of mRNA for these and other melanocyte lineage antigens.

120 animals total per study:

15 animals receiving only MU-X tumor and injected with saline only onday 0. Tumor will be excised daily from 5 animals for in vitro assay ofantigen expression at days 1, 3 and 7.

-   -   45 animals receiving MU-X tumor followed by intravenous        injection of IFN-β on day 0 at 3 dosage levels (10, 100, and        1000 IU/g animal weight). Tumor will be excised from 5 animals        in each dosage group for in vitro assay of antigen expression at        days 1, 3 and 7.

15 animals receiving only MU tumor and injected with saline only on day0. Tumor will be excised daily from 5 animals for in vitro assay ofantigen expression at days days 1, 3 and 7.

-   -   45 animals receiving MU tumor followed by intralesional        injection of human IFN-β on day 0 at 3 dosage levels (10, 100,        and 1000 IU/g animal weight). Tumor will be excised from 5        animals in each dosage group for in vitro assay of antigen        expression at days 1, 3 and 7.

1-65. (canceled)
 66. A method of treating a tumor comprising administering to a subject with a tumor an amount of interferon-β (IFN-β) receptor agonist to up-regulate expression of a tumor associated antigen (TAA) on the tumor followed by administering an autologous immune cell that interacts with a tumor cell of the tumor, wherein the IFN-β receptor agonist comprises a polypeptide comprising the amino acid sequence of SEQ ID NO: 29 or SEQ ID NO: 30, thereby treating the tumor.
 67. The method of claim 66, wherein the immune cell is a lymphocyte.
 68. The method of claim 67, wherein the lymphocyte is a T lymphocyte.
 69. The method of claim 66, wherein the amino acid sequence of the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:
 29. 70. The method of claim 69, wherein the amino acid sequence of the polypeptide consists of the amino acid sequence set forth in SEQ ID NO:
 29. 71. The method of claim 66, wherein the amino acid sequence of the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:
 30. 72. The method of claim 71, wherein the amino acid sequence of the polypeptide consists of the amino acid sequence set forth in SEQ ID NO:
 30. 73. The method of claim 66, wherein the tumor is metastatic.
 74. The method of claim 66, wherein the treatment reduces tumor volume, inhibits an increase in tumor volume, stimulates tumor cell lysis or apoptosis, or reduces tumor metastasis.
 75. The method of claim 66, further comprising administering an anti-tumor therapy.
 76. The method of claim 75, wherein the anti-tumor therapy comprises surgical resection, radiotherapy, or chemotherapy.
 77. The method of claim 66, wherein the subject is human.
 78. The method of claim 66, wherein the TAA is selected from: Melan-A/MART-1, tyrosinase, gp100/pmel 17, TRP-1, TRP-2, an MITF, MITF-A, MITF-M, melanoma GP75, Annexin I, Annexin II, adenosine deaminase-binding protein (ADAbp), PGP 9.5, Colorectal associated antigen (CRC)—C017-1A/GA733, Ab2 BR3E4, CI17-1A/GA733, Hsp70, Hsp90, Hsp96, Hsp105, Hsp110, HSPPC-96, stress protein gp96 (a human colorectal cancer tumor rejection antigen, Heike 2000), gp96-associated cellular peptide, G250, Dipeptidyl peptidase IV (DPPIV), Mammaglobin, thyroglobulin, STn, Carcinoembryonic Antigen (CEA), Carcinoembryonic Antigen (CEA) epitope CAP-1, Carcinoembryonic Antigen (CEA) epitope CAP-2, etv6, aml1, Prostate Specific Antigen (PSA), PSA epitope PSA-1, PSA epitope PSA-2, PSA epitope PSA-3, Ad5-PSA, prostate-specific membrane antigen (PSMA), Prostatic Acid Phosphatase (PAP), Prostate epithelium-derived Ets transcription factor (PDEF), Parathyroid-hormone-related protein (PTH-rP), EGFR, PLU1, Oncofetal antigenimmature laminin receptor (OFA-iLR), MN/CA IX (CA9) (Shimizu, 2003), HP59, Cytochrome oxidase 1, sp100, msa, Ran GTPase activating protein, a RabGAP (Rab GTPase-activating) protein, PARIS-1, T-cell receptor/CD3-zeta chain, cTAGE-1, SCP-1, Glycolipid antigen-GM2, GD2 or GD3, GM3, FucosylGM1, Glycoprotein (mucin) antigens-Tn, Sialyl-Tn, TF and Mucin-1, CA125 (MUC-16), a MAGE family antigen, GAGE-1,2, BAGE, RAGE, LAGE-1, GnT-V, EPCAM/KSA, CDK4, a MUC family antigen, HER2/neu, ErbB-2/neu, p21 ras, RCAS1, α-fetoprotein, E-cadherin, α-catenin, β-catenin and γ-catenin, NeuGcGM3, Fos related antigen, Cyclophilin B, RCAS1, S2, L10a, L 10a, Telomerase rt peptide, cdc27, fodrin, p120ctn, PRAME, GA733/EoCam, NY-BR-1, NY-BR-2 NY-BR-3, NY-BR-4 NY-BR-5, NY-BR-6 NY-BR-7, NY-ESO-1, L19H1, MAZ, PINCH, PRAME, Prp1p/Zer1p, WT1, adenomatous polyposis coli protein (APC), PHF3, LAGE-1, SART3, SCP-1, SSX-1, SSX-2, SSX-4, TAG-72, TRAG-3, MBTAA, a Smad tumor antigen, lmp-1, HPV-16 E7, c-erbB-2, EBV-encoded nuclear antigen (EBNA)-1, Herpes simplex thymidine kinase (HSVtk), alternatively spliced isoform of XAGE-1 (L552S), TGF beta RII frame shift mutation, BAX frame shift mutation, or an antigenic fragment thereof.
 79. A method of treating a subject having or at risk of having a tumor comprising administering to the subject a) an amount of interferon-β (IFN-β receptor agonist that up-regulates expression of a tumor associated antigen (TAA) on the tumor and b) the TAA, wherein the TAA is administered singly or multiple times to the subject 1 day to 6 months before administering the IFN-β receptor agonist, and wherein the IFN-β receptor agonist comprises a polypeptide comprising the amino acid sequence of SEQ ID NO: 29 or SEQ ID NO: 30, thereby treating the subject.
 80. The method of claim 79, wherein the amino acid sequence of the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:
 29. 81. The method of claim 80, wherein the amino acid sequence of the polypeptide consists of the amino acid sequence set forth in SEQ ID NO:
 29. 82. The method of claim 79, wherein the amino acid sequence of the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:
 30. 83. The method of claim 82, wherein the amino acid sequence of the polypeptide consists of the amino acid sequence set forth in SEQ ID NO:
 30. 84. The method of claim 79, wherein the IFN-β receptor agonist is administered singly or multiple times.
 85. The method of claim 79, wherein the TAA is administered singly or multiple times to the subject 1 to 14 days before administering the IFN-β receptor agonist.
 86. The method of claim 79, wherein the TAA is administered singly or multiple times to the subject 14 to 30 days before administering the IFN-β receptor agonist.
 87. The method of claim 79, wherein the TAA is administered singly or multiple times to the subject 1 to 6 months before administering the IFN-β receptor agonist.
 88. The method of claim 79, wherein the tumor is metastatic.
 89. The method of claim 79, wherein the treatment reduces tumor volume, inhibits an increase in tumor volume, stimulates tumor cell lysis or apoptosis, or reduces tumor metastasis.
 90. The method of claim 79, wherein the treatment reduces one or more adverse symptoms associated with the tumor.
 91. The method of claim 79, wherein the treatment inhibits progression of the tumor.
 92. The method of claim 79, wherein the subject is a candidate for, is undergoing, or has undergone anti-tumor therapy.
 93. The method of claim 92, wherein the anti-tumor therapy comprises surgical resection, radiotherapy, or chemotherapy.
 94. The method of claim 79, further comprising administering an autologous immune cell that interacts with a cell of the tumor.
 95. The method of claim 94, wherein the immune cell is a lymphocyte.
 96. The method of claim 95, wherein the lymphocyte is a T lymphocyte.
 97. The method of claim 79, wherein the subject is human.
 98. The method of claim 79, wherein the TAA is selected from: Melan-A/MART-1, tyrosinase, gp100/pmel 17, TRP-1, TRP-2, an MITF, MITF-A, MITF-M, melanoma GP75, Annexin I, Annexin II, adenosine deaminase-binding protein (ADAbp), PGP 9.5, Colorectal associated antigen (CRC)—C017-1A/GA733, Ab2 BR3E4, CI17-1A/GA733, Hsp70, Hsp90, Hsp96, Hsp105, Hsp110, HSPPC-96, stress protein gp96 (a human colorectal cancer tumor rejection antigen, Heike 2000), gp96-associated cellular peptide, G250, Dipeptidyl peptidase IV (DPPIV), Mammaglobin, thyroglobulin, STn, Carcinoembryonic Antigen (CEA), Carcinoembryonic Antigen (CEA) epitope CAP-1, Carcinoembryonic Antigen (CEA) epitope CAP-2, etv6, aml1, Prostate Specific Antigen (PSA), PSA epitope PSA-1, PSA epitope PSA-2, PSA epitope PSA-3, Ad5-PSA, prostate-specific membrane antigen (PSMA), Prostatic Acid Phosphatase (PAP), Prostate epithelium-derived Ets transcription factor (PDEF), Parathyroid-hormone-related protein (PTH-rP), EGFR, PLU1, Oncofetal antigenimmature laminin receptor (OFA-iLR), MN/CA IX (CA9) (Shimizu, 2003), HP59, Cytochrome oxidase 1, sp100, msa, Ran GTPase activating protein, a RabGAP (Rab GTPase-activating) protein, PARIS-1, T-cell receptor/CD3-zeta chain, cTAGE-1, SCP-1, Glycolipid antigen-GM2, GD2 or GD3, GM3, FucosylGM1, Glycoprotein (mucin) antigens-Tn, Sialyl-Tn, TF and Mucin-1, CA125 (MUC-16), a MAGE family antigen, GAGE-1,2, BAGE, RAGE, LAGE-1, GnT-V, EPCAM/KSA, CDK4, a MUC family antigen, HER2/neu, ErbB-2/neu, p21ras, RCAS1, α-fetoprotein, E-cadherin, α-catenin, β-catenin and γ-catenin, NeuGcGM3, Fos related antigen, Cyclophilin B, RCAS1, S2, L10a, L10a, Telomerase rt peptide, cdc27, fodrin, p120ctn, PRAME, GA733/EoCam, NY-BR-1, NY-BR-2 NY-BR-3, NY-BR-4 NY-BR-5, NY-BR-6 NY-BR-7, NY-ESO-1, L19H1, MAZ, PINCH, PRAME, Prp1p/Zer1p, WT1, adenomatous polyposis coli protein (APC), PHF3, LAGE-1, SART3, SCP-1, SSX-1, SSX-2, SSX-4, TAG-72, TRAG-3, MBTAA, a Smad tumor antigen, lmp-1, HPV-16 E7, c-erbB-2, EBV-encoded nuclear antigen (EBNA)-1, Herpes simplex thymidine kinase (HSVtk), alternatively spliced isoform of XAGE-1 (L552S), TGF beta RII frame shift mutation, BAX frame shift mutation, or an antigenic fragment thereof. 